FLAVONOIDS AND STEROID HORMONE-DEPENDENT CANCERS

Rachel Stacey Rosenberg Zand

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Nutritional Sciences University of Toronto

O Copyright by Rachel Stacey Rosenberg Zand 2001 National Library Bibliothèque nationale of Canada do Canada Aquisiîions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. nie Wellington Onam ON KIA ON4 ûüawa ON KIA ON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive Licence allowing the exciusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distriiuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. Ia 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 ihèse. thesis nor substantial extracts kom 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, FLAVONOIDS AND STEROID HORMONE-DE PEND EN^ CANCERS DOCTOR OF PHILOSOPHY 2001 RACHELSTACEYROSENBERGZAND GRADUATE DEPARTMENT OF NUTRITIONAL SCIENCES UNIVERSITY OF TORONTO

ABSTRACT

Steroid hormone-dependent cancers, including breast and prostate, are leading causes of cancer morbidity and mortality in North America. In Asian countries, these diseases are much less common. Nutritional and lifestyle factors are associated with differences in incidence, with an emphasis on consumption of plant foods, in particular soy. Flavonoids are one class of compounds present in these plant foods. With over

4000 identified members, these compounds have been hypothesized to have several important chemopreventive effects.

The purpose of this project was to assess the steroid horrnone activities of plant foods, with a reference to soy, in three forms: pure flavonoids, plant extract

(nutraceutical), and whole (functional) food. After developing an in vitro bioassay, soy isoflavones and other flavonoids were evaluated for steroid hormone activities, defined as their ability to induce or inhibit production of estrogen- (pS2) and androgedprogestin-

(prostate-specific antigen, PSA) regulated proteins. Genistein and biochanin A demonstrated highest estrogenic activity of the compounds tested, which was dose- response down to 10~M and 10" M, respectively. These soy isoflavones slso demonstrated significant anti-andmgen activity (76% and 98%, respectively at 10" M).

Natural products and nutraceuticals were then tested for steroid horrnone activities. As with pure isoflavones, the commercial soy and red clover extracts had high estrogenic activity. Soy extract demonstrated 63% ariti-androgen activity at the highest strength testeci.

Finally, we determined the steroid hormone potentials of one functional food, soy, in healthy subjects. By using biological fluids obtained from individuals in a soy feeding study, we found that short-term feeding did not increase agonist potentials. Moreover, non-significant reductions in estrogenic and androgenic activities in women and men, respectively, may indicate that long-term feeding of this functional food rnay reduce risk factors for steroid-hormone dependent cancers.

These results indicate that soy isoflavones and other flavonoids havè steroid hormone activities. These activities may act in vivo to modulate activities of potentially carcinogenic endogenous hormones. Further research is needed to determine the phyçiological importance of flavonoids in prevention andior management of homone- dependent cancers, and how to make best use of natural products, nutraceuticals and functional foods in individuals at risk. ACKNOWLEDGEMENTS

I would like to first and foremost acknowledge and thank my supervisors, Drs.

David Jenkins and Eleftherios Diamandis, for their mentorship, assistance and patience with me over the past 5 years. They have taught me many lessons, both scientific and life, and will remain major influences in my life's path. Thank you to my advisors Drs.

Venket Rao and Tibor Heim, for always being there when I needed their guidance, or just their ear.

Thank you to Linda Grass, Antoninus Soosaipillai and Dr. Bhupinder Bharaj for their technical assistance and friendship throughout my PhD project, and to my many labmates who have supported me throughout this process: Christina Obiezu, George

Yousef, Angeliki Magklara, Liu-Ying Luo, Albert Chang, Jenny Vassilikos and Hani Kim.

Thanks to Ed Vidgen for the biostatistical analysis for Chapter 5.

I would like to thank my parents Dr. David and Phyllis Rosenberg for their emotional support throughout my university education. I hope that 1 can continue to make them proud.

And finally, to my husband, Frank Zand, thank you for putting up with al1 my day- to-day worries, concems, joys, and frustrations that made up this experience. May we share only successes in the years to come. TABLE OF CONTENTS

ii

Acknowledgements iv

List of Abbreviations viii

List of Tables xi

List of Figures xii

List of Appendices xiii

List of Publications Arising from this Work xiv

Chapter 1: lntroduction and Literature Review 1 1 .l lntroduction 2 1.2 Literature Review . 3 1.2.1 Flavonoids and Steroid Hormone-Dependent Cancers 3 1.2.1.1 lntroduction 3 1.2.1.2 Epidemiological Evidence 8 1.2.1.3 In Vitro and Animal Models 1O 1 -2.1.4 Influences on Modulators of Risk for Steroid Hormone-Dependent Cancers 33 1.2.1.5 Discussion 38 1.2.2 Steroid Hormone Receptors 39 1.2.3 Steroid Hormone Receptor Signaling 41 1.3 Hypothesis 42 1.4 Objectives 42

Chapter 2: Development of Tools to Measure Steroid Hormone Activity 44 2.1 Rationale 45 2.2 Developrnent and Evaluation of a Competitive Tirne-Resolved Iminunofluorometric Assay for the Estrogen-Regulated Protein pS2 46 2.2.1 Introduction 46 2.2.2 Materials and Methods 47 2.2.2.1 Instrumentation 48 2.2.2.2 pS2 Standard Solutions 49 2.2.2.3 Procedures 49 2.2.2.4 Tissue Culture Experiments with Cell Lines 50 2.2.3 Results 51 2.2.3.1 Antibody Selection and Assay Optimization 51 2.2.3.2 Calibration Curve, Detection Limit and Precision 52 2.2.3.3 Tissue Culture Experiments 52 2.2.4 Discussion 52

2.3 Is ICI 182,780 an Anti-Progestin in Addition to being an Anti-Estrogen? 57 2.3.1 Introduction 57 2.3.2 Materials and Methods 60 2.3.2.1 Materials 60 2.3.2.2 Methods 60 2.3.3 Results 62 2.3.4 Discussion 69

Chapter 3: Steroid Hormone Activities of Flavonoids 73 3.1 Rationale 74 3.2 Agonist Activity of Flavonoids and Related Compounds on Steroid Hormone Receptors 3.2.1 Introduction 3.2.2 Materials and Methods 3.2.2.1 Materials 3.2.2.2 Methods 3.2.3 Results 3.2.4 Discussion 3.3 Genistein: A Potent Naturat Anti-androgen" 3.4 Flavonoids Can Block PSA Production by Breast and Prostate Cancer Cell Lines 3.4.1 Introduction 3.4.2 Materials and Methods 3.4.2.1 Materials 3.4.2.2 Methods 3.4.3 Results 3.4.4 Discussion

Chapter 4: Steroid Hormone Activity of Natural Products and Nutraceuticals 4.1 Rationale 4.2 Effects of Natural Products and Nutraceuticals on Steroid Hormone- Regulated Gene Expression 1O5 4.2.1 Introduction 105 4.2.2 Materials and Methods 106 4.2.2.1 Materials 106 4.2.2.2 Method 107 4.2.3 Results 111 4.2.4 Discussion 114 Chapter 5: Analyses of In Vivo Effects of Functional Foods 5.1 Rationale 5.2 Effects of Soy Protein Foods on Low-Density tipoprotein Oxidation and Ex Vivo Sex Hormone Receptor Activity 5.2.1 Introduction 5.2.2 Subjects and Methods 5.2.2.1 Subjects 5.2.2.2 Methods 5.2.3 Results 5.2.3.1 Urinary lsoflavone Excrefion 5.2.3.2 Ex Vivo Sex Hormone Receptor Activity 5.2.4 Discussion

Chapter 6: General Discussion and Conclusions 6.1 General Discussion 6.2 Summary 6.3 Future Directions 6.4 Conclusion

Chapter 7: References

Appendix LIST OF ABBREVIATIONS

Abbreviation Full Name

Ab Antibody

AF Activation function

AIN American lnstitute of Nutrition

AOM Azoxymethane

AR Androgen receptor

ARE Androgen response element

BaP Benro(a)pyrene

BSA Bovine serum antigen

CAT Chloramphenicol acetyl transferase

COX Cyclooxygenase

cDNA Complementary deoxyribonucleic acid

DES Diethylstilbestrol

DHT Dihydrotestosterone

DMAB 3,2'-Dirnethyl-4-aminobiphenyl

DMBA 7,12-Dimethylbenz[a]anthracene

ECG Epicatechin gallate

EGCG Epigallocatechin gallate

EGF Epithelial growth factor

ELISA Enzyme-linked immunosorbent assay

ER Estrogen receptor ERE Estrogen response element

F Fischer

FAD Focal area of dysplasia

GCDFP Gross cystic disease fluid protein

GR Glucocorticoid receptor

GRE Glucocorticoid response element

HDL-C High density lipoprotein cholesterol hK2 Human glandular kallikrein 2

HRE Hormone response element

170-HSD 170-H ydroxysteroid dehyd rogenase

170-HSOR 17P-Hydroxysteroid oxidoreductase

HSP Heat shock protein

IGF-1 Insulin-like growth factor

1 p3 lnositol 1,4,5-triphosphate luc Luciferase

M Molar

MAP Mitogen activated protein

NAF Nipple aspirate fluid

NMU Nitrosomethylurea

PCR Polymerase chah reaction

PhlP 2-Amino-1-methylS-phenylimidazo[4,5-

blpyridine

Progesterone receptor PRE Progesterone response element

PSA Prostate specific antigen

RIA Radiometric imunoassay

RAR Retinoic acid receptor

RXR Retinoic acid receptor

SAMlg Sheep anti-mouse immunoglobulin G

SD Sprague Dawley

SPI Soy protein isolate

TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin

TE0 Terminal end buds

TG F-B Transforrning growth factor

TPA 12-0-Tetradecanoyl phorbol 13-acetate LIST OF TABLES

Table 1.1 Food Sources and Structures of Specific Flavonoids

Table 1.2 Cell Lines Used

Table 1.3 Cell Cycle

Table 2.1 Production of pS2, PSA and hK2 by Blockers and Steroids

Table 3.1 Flavonoids and Related Compounds With and Without Steroid Hormone Agonist Activity

Table 3.2 F!avonoids and Related Compounds that Significantly Inhibited PSA Production in the 8T-474 and PC-3(AR)2Cell Lines

Table 3.3 Flavonoids and Related Compounds That Did Not lnhibit PSA Production

Table 4.1 Natural Products and Nutraceuticals Tested

Table 4.2 Biological Activity of Natural Products as Detected by ouf Tissue Culture Screening Procedure

Table 5.1 Caiculated Macronutrient lntake (mean + SE) on the Test and Control Metabolic Diets (N=31)

Tabte 5.2 tsoflavone Content of Study Diets 123 LIST OF FIGURES

Figure Structures of Flavonoids

Figure Structures of Steroid Hormones and Antagonists

Figure Regulation of Steroid Honnone-Regulated Genes

Figure 1.4 Main Biosynthetic and lnactivating Pathways of Estrogens and Androgens

Figure 1.5 Signal Transduction Pathways lnfluenced by Flavonoids

Figure 2.1 Typical Calibration Curve for the pS2 Competitive Assay

Figure 2.2 pS2 Production by Six Steroids and Alcohol

Figure 2.3 Time Course of pS2 Production by Estradiol

Figure 2.4 Dose-Response of Steroids at Concentrations between IO*' - 10-l2M Figure 2.5 Structures of ICI 182,780 (faslodex) and RU 486 (mifepristone) figure 2.6 Percent Blocking of pS2 Production, by the Anti-estrogen ICI 182,780 and Anti-progestin RU 486

Figure 2.7 Percent Blocking of Human Glandular Kallikrein 2 (hK2) Production by ICI 182,780 and RU 486

Figure 2.8 Percent Blocking of hK2 Production by ICI 182,780 and RU 486

Figure 2.9 Percent Blocking of Prostate-Specific Antigen (PSA) Production by ICI 182,780 and RU 486

Figure 2.1 0 Percent Blocking of PSA Production by ICI 182,780 and RU 486

Figure 3.1 Estrogenic Activity of Three Isoflavones, Measured as ng of pS2 proteinhnL in the Tissue Culture Supernatant

Figure 3.2 Percent Blocking of DHT as Measured by PSA Production by Genistein, Quercetin and Nilutamide 9 1 Figure 3.3 Dose-Response Inhibition of PSA Production of Flavonoids and Related Compounds in the BT-474 Human Breast Cancer Cell Line 100

Figure 4.1 Dose-Response Estrogen Activity of PromensilTMand Estro-LogicTMt 12

Figure 5.1 Creatinine-Corrected Urinary Estrogen and Androgen Equivalents in Men an Postmenopausal Woment on Test and Control Diets 128

LIST OF APPENDICES

Appendix I Structures of Flavonoids and Related Compounds LIST OF PUBUCAflONS ARISING FROM THIS WORK

Rosenberg Zand RS, Jenkins DJA, Diamandis €P. Flavonoids and steroid hormone-dependent cancers: a review. J C hromatograp hy 2001 (submitted).

Rosenberg Zand RS, Jenkins DJA, Diamandis €P. Effeds of natural products and nutraceuticals on steroid homone-regulated gene expression. Clinica Chimica Acta 2001 (submitted).

Rosenberg Zand RS, Jenkins DJA, Brown, TJ, Diamandis EP. Fiavonoids can block PSA production in breast and prostate cancer cell lines. Clinica Chimica Acta 2001 (in press).

Jenkins DJA, Kendall CWC, Popovich DG, Vidgen E, Mehling CC, Vuksan V, Ransom TPP, Rao AV, Rosenberg Zand RS et al. Effect of a very-high-fiber vegetable, fruit, and nut diet on serum lipids and cotonic function. Metabotism 2001 ;50:494-503.

Rosenberg Zand RS, Jenkins DJA, Diamandis €P. Agonistic activity of Ravonoids and related compounds on steroid hormone receptors. Breast Cancer Res Treat 2000;62:35-49.

Rosenberg Zand RS, Jenkins DJA, Diamandis EP. Genistein: a potential natural anti-androgen. Clin Chem 2000;46:887-8.

Rosenberg Zand RS, Grass L, Magklara A, Jenkins DJA, Diamandis €P. Is ICI 182,780 an anti-progestin in addition to being an anti-estrogen? Breast Cancer Res Treat 2000;60:1-8.

Jenkins DJA, Kendall CWC, Mehling C, Agarwal S, Rao AV, Rosenberg Zand RS, Diamandis EP et ai. Effect of soy protein foods on low density lipoprotein cholesterol oxidation and ex vivo sex homone receptor activity - a controlled crossover trial. Metabolism 2000;49:537-43.

Rosenberg Zand RS, Diamandis €P. Developrnent and assessrnent of a competitive assay for the estrogen-regulated pS2 protein. J Clin Lab Analysis 1999; l3(5):241-245.

Jenkins DJA, Kendall CWC, Vidgen E, Agamal S, Rao AV, Rosenberg RS, Diamandis EP, Novokmet R, Mehling CC, Perera Tl Griffm LC, Cunnane SC. Health aspects of partially defatted flaxseed: effects on serum Iipids, oxidative rneasures and ex vivo androgen and progestin activities - a controlled crossover trial. Am J Clin Nutr 1999;69:395-402.

xiv Posters

1. RS Rosenberg Zand, DJA Jenkins, EP Diamandis. Steroid Hormone Effects of Natural Products and Nutraceuticals. American Association for Cancer Research (Abstract #4667). New Orleans, LA, March 24-28,2001.

2. RS Rosenberg Zand, DJA Jenkins, EP Diamandis. Anti-androgen Activity of Flavonoids in Two Human Cancer Cell Lines. Inter-Provincial Graduate Nutrition Exchange. Toronto, ON, June 8-9,2000.

3. RS Rosenberg Zand, DJA Jenkins, EP Diamandis. Anti-androgen Activity of Flavonoids. Canadian Society of Clinical Chemistry. Hamilton, ON, June 3-7, 2000.

4. RS Rosenberg Zand, DJ Jenkins, EP Diamandis. Antagonist Steroid Hormone Activity of Flavonoids and Related Compounds in an In Vitro Tissue Culture System. American Association for Cancer Research (Abstract #5377), San Francisco, CA, April 1-5,2000.

5. RS Rosenberg Zand, DJA Jenkins, EP Diamandis. Agonistic Steroid Hormone Activity of Flavonoids and Related Compounds in an In Vitro Tissue Culture System. American lnstitute of Cancer Research, Washington DC, September 2- 3, 1999. CHAPTER 1: INTROOUCflON AND LITERATURE REVIEW CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Hormone-dependent cancers, including those of the breast and prostate, are leading causes of cancer morbidity and rnortality of women and men in North America

(1). Current medical therapies focus on hormone treatrnent, whether it be hormone antagonists, e-g. antiestrogens; strong hormone agonists, e.g. progestin derivatives; or drugs aimed at inhibiting the formation of more carcinogenic forms of hormones, e.g.

Sa-reductase inhibitors to block production of dihydrotestosterone. Alternative therapies have looked towards natural products including foods and herbs to combat, or complernent treatments of these diseases. Western medicine originally dismissed such alternatives as quackery. However, researchers have discovered that several plants and plant foods contain compounds very similar in structure to the anti-hormones and hormones given in pharmaceutical treatments, as well as endogenous steroid hormones. One group of these compounds are the flavonoids, a class of over 4000 members which are concentrated in plant foods, including fruits, vegetabtes, legumes, red wine and tea.

Although many epidemiological studies have related consumption of soy isoflavones to lower risks of steroid hormone-dependent cancers, very little is known about the mechanisms by which these reductions of risk occur. Therefore, the purpose of this research was to assess one mechanism by which flavonoids, in general, and soy isoflavones, specifically, may reduce incidences of these cancers. Through an in vitro bioassay, the steroid hormone activities of flavonoids were detemined: as pure compounds, as extracts of natural products and nutraceuticals, and finally, as biological fluids from subjects on soy feeding studies. These results may provide further insight into how flavonoids can be used in the prevention andior management of steroid hormone-dependent cancers.

1.2 Literature Review

1.2.1 Flavonoids and Steroid Hormonedependent Cancers

1.2.1.1 Introduction

Steroid- hormone dependent cancers, including t hose of the breast, prostate and colon, are leading causes of morbidity and mortality in western countries. In Asian countries, particulariy rural areas, these diseases are relatively uncornmon.

Epidemiological studies contribute these differences in incidence to environmental causes, particulariy those of diet and Iifestyle. Dietary factors include low consumption of fruit, vegetables and legurnes, particularty soy, in the west. Flavonoids are one component of these plant foods being investigated for their role in chemoprevention.

Flavonoids are phenolic compounds characterized by their diaryl nucleus (Figure

1.1). They are structurally similar to steroid hormones (Figure 1.2), particularly estrogens, and therefore have been studied over the past several years for their potential roles in prevention of hormone-dependent cancers. Over 4000 flavonoids have been characterized in edible plant foods, and are consumed in highest amounts in soy, apples, red wine, tea and onions (Table 1.1)(1). Human consurnption of al1 flavonoids has been estimated tu be a few hundred milligrams to 1 g per day (2). Flavones Flavanones Isoflavones

Flavans

Figure 1.1. Structures of Flavonoids. General structures of flavones, isoflavones, flavanones and flavans. 17B-Estradiol Testosterone Progesterone

Tamoxifen ICI t 82,780

Mifepristone (RU 486)

Figure 1.2. Structures of Steroid Hormones and Antagonists Table 1.1. Food Sources and Structures of Specific Flavonoids

Food Source Structure

Satechin Tea

Coumestrol SOY

Daidzein Soy, other legumes Genistein Soy, other legumes

Hesperetin Orange, lemons

Broccoli, tea Naringenin Orange, grapefruit

Quercetin Onion, broccoli, red wine, tea

This review shall present the current knowledge and research available in the

field of flavonoids and steroid hormone-dependent cancers. Several mechanisms will

be discussed, from associations made through epidemiology, through in vitro studies to

animal models and human trials. Much work is still needed to firmly establish the

chemopieventive applications of flavonoids, and the foods in which they are contained-

1.2.1.2 E~idemioloaicalEvidence

The original associations between flavonoids and steroid hormone-dependent

cancers were made through several ecological studies looking at the low incidences of

breast, colon and prostate cancers in Asian countries compared to western ones. Soy

consumption is very high in Asia compared to North America and Europe, and it was

found that this food could be negatively correlated with cancer incidence (3-5). Since these initial studies, many other plant foods have been studied for their associations with these cancers.

Most epiderniological studies have concentrated on whoie foods, not components present within them. Through these studies, many interesting associations have been made between vegetable consumption and various cancers. Messina et al have extensively reviewed the in vivo and in vitro data of soy, showing protective effects in both animal rnodels and epidemiologic studies (6). Steinmetz and Potter (7) have reviewed over 200 case-controi and cohort studies, finding that over the majority of those investigating cancers of the breast, colon, cervix, endometriurn and ovary have shown statistically significant decreases in incidence with vegetable and/or fruit consumption (8-60). The only site for which such a relationship was not found was the prostate (60-64). However, a Seventh-day Adventist study, involving over 34,000 vegetarians and omnivores, found that vegetarians had sig nificantly reduced risks of colon and prostate cancer rates.

Epidemiological studies looking specifically at certain flavonoids are becoming more important, as databases and techniques for quantifying consumption of these compounds are improving (61-66). Flavonoids present in highest amounts in the human diet include the soy isoflavones genistein, daidzein and biochanin A, flavonols quercetin, myricetin and kaempferol, and the flavones luteolin and apigenin (2,fl). One case-control study used this new database (70) to look at 83 prostate cancer patients and 107 age-matched controls. They found that after adjustments for total calories, greater consumption of most phytoestrogens, induding isoflavones and other flavonoids, had a slightly protective effect on prostate cancer risk. Moreover, genistein, daidzein, and coumestrol (a sttucturally-related compound) showed the strongest protective associations. However, the overall intake of the isoflavones was very low compared to Japanese men (1.2 mg versus 39.2 mg/day). Some studies have reported weak protective effects of flavonoids from fruits and vegetables and prostate and colon cancers (71,72). Others have shown no associations between flavonoid intake and cancer mcirtality or incidence (73,741. At present, relationships between whole foods,

Le. vegetables and soy, show much stronger chemopreventive associations than the flavonoids that they contain.

1.2.1.3 In Vitro and Animal Models

Over the last few years, various mechanisms by which flavonoids may prevent, or

even treat steroid hormone-dependent cancers, have been investigated. These include

activities at the tissue, cellular (Table 1.2), and even sub-cellular levels. We wili

concentrate here on steroidal activities, proliferation, antioxidant activities, signal

transduction, and tumorigenic pathways.

Estroaenic Activitv

Estrogenic effects of flavonoids were first discovered through determining the

cause of infertility in Australian sheep eating red clover. The leaves contain up to 5% by

dry weight of the soy isoflavones biochanin A and formononetin (75,76). Of the

flavonoids, the soy isoflavone genistein has remained the model "phytoestrogenn.

Genistein is structurally similar to estradiol, other steroid hormones (testosterone, Table 1.2. Cell Lines Used

1 TYP~ 1 Cell Line 1 Steroid~Receptor 1 Expression Breast MCF-7 ER, PR (+) BT-474 ER, PR, AR (+) T47-D ER, PR, AR (+) MDA-MB-468 ER, PR (-) Prostate LNCaP AR (+) VeCaP AR (-) PC-3 AR (-) Colon HT-29 none (VDR (+)) Caco-2 none Ovary , OVCAR-5 ER(+)

progesterone), and synthetic inhibitors including tamoxifen. This isoflavone possesses

111000 the estrogenic activity of estradiol (77-79). Circulating concentrations in

individuals consuming moderate amounts of soybeans are nearly 1000-fold higher than

peak levels of endogenous estradiol in premenopausal women (80-82). Genistein and

other soy isoflavones have been shown to compete with radiolabeled estradiol for

binding to the estrogen receptor (83). This may be because of the structural similarity

between the steroid nucleus of 17P-estradiol and the polyphenolic ring structure of

isoflavones. Since other plant-derived flavonoids also possess this structure, they too

have become relatively well-studied for estrogenic and anti-estrogenic activities. In

contrast, androgenic activities of flavonoids have only recently started being

investigated. The data obtained from these studies support important applications for

these cornpounds and the foods in which they are found, in prostate cancer prevention.

Steroid hormone activities are important because they are mechanisms by which

ceils cornmunicate with their environment. Direct agonistic activities consist of, in

simplified ternis, a ligand (estrogen, E or androgen, A) binding to a nuclear receptor (estrogen, ER or androgen, AR), resulting in the dimerization of the nuclear receptor.

This ligand-nuclear receptor complex then binds to a hormone response element (HRE, either estrogen, ER€ or androgen, ARE) on estrogen- or androgen-dependent genes, respectively, leading to its transcription. The mRNAs are then translated, and proteins are produced, exerting biological responses (Figure 1.3). Beato et al describe this mechanisrn in much greater detail (84). The effects of steroid hormones in the carcinogenesis process are very complex. Ovariectomy is the oldest treatment for breast cancer (85). Surgical castration is still an option for prostate cancer (86), and chernical castration is a common strategy for managing its aggressiveness today (87-

89). Timing of estrogens may also be important. Some studies indicate that while exposure to diethylstilbestrol (DES) in utero may predispose children to adult cancers several decades later (90-94), prepubertal estrogens rnay be protective (95). During adulthood, these hormones are once again linked to carcinogenicity .

The steroid hormone pathways described above are utilized in the development of assays to determine the agonistic and antagonist steroid hormone activities of compounds. This is accomplished either through measurement of estrogen-or androgen-regulated proteins, reporter proteins such as chloramphenicol aceîyl Steroid

Endo~lasmicReticulum

Cytoplasm

Figure 1.3. Regulation of Steroid Hormone-Regulated Genes. When a ligand diffuses into the cell, it will bind to its respective steroid hormone receptor in the nucleus of the cell. The receptor is then activated through dimerization and phosphorylation, and the steroid horrnone- steroid hormone receptor complex may then bind to a hormone response element (HRE) in the promoter region of a steroid hormone-dependent gene. The gene is then transcribed to mRNA, and further translated to protein. This protein may remain in the cell, or may be secreted into the supernatant surrounding the cell. Several techniques are currently used to measure steroid hormone activity, based upon this mechanism. transferase (CAT) and luciferase, with ERE or ARE promoters, or Southern (using cDNA) and Northem blots.

Zava et al have determined estrogenic and anti-estrogenic activities of several flavonoids, including genistein, equol, kaempferol and hesperetin using two rnethods in

ER(+) and ER(-) breast cancer cell lines (96). The first involved incubating ER(+) cells

(MCF-7) in tubes containing both ['251]estradiol and flavonoid for 5 minutes at 4' C, aspirating the supernatant, and measuring the r2s11estradiol remaining in the nuclei,

using a gamma counter. Results were then expressed as percent ['251]estradiol binding to ER in the nucleus in the absence or presence of increasing concentration of flavonoid, from 100 pM to 10 FM. This technique demonstrated specific binding of test compound to ER, and thereby, anti-estrogenic activity. The second technique, that to

rneasure estrogenic activity, involved measuring the estrogen-regulated, secreted

protein, pS2, after incubating the cells with flavonoid at the above concentrations for 72

hours. The assay used here was a two-site enzyme-linked immunosorbent assay.

Genistein and equol, the metabolite of the soy isoflavone daidtein, were found fo have

the highest binding affinities, 0.1 that of estradiol. Next came kaempferol (0.012), and

finally quercetin (0.001). Hesperetin and p-naphthoflavone did not have blocking

activity. All of the compounds found to bind to the ER in the previous study stimulated

pS2 production, in the same order as their binding activity. Blocking of pS2 production

by tamuxifen further confirmed that binding to the ER was the mechanism observed.

Similar results for genistein and other flavonoids were seen through measuring pS2

andior cathepsin-D transcription (83,97-99). Our group (100) has evaluated estrogenic

activity of over 70 flavonoids and structurally-related compounds using $32 concentrations in the supernatant as the endpoint. Structure-function relationships were discussed in this study, with the diphenolic structure, and positions of hydroxyl groups residing in the C-7 or 4' positions (Figure 1.1) being most important for estrogenic activity. Flavonoids with hydroxyl groups at the C-3 position, such as quercetin, hinders binding, as is reflected by the low estrogeniclbinding affinity in both studies. At least two other studies, using different systems, concur with these resuits. Miksicek assessed estrogenicity of flavonoids (at 1 PM) using HeLa cells transfected with wild- type, recombinant estrogen cDNA expressed from the plasmid pER-18, and an estrogen-responsive reporter plasmid system measuring production of CAT (pERE-TK-

CAT). Genistein displayed highest activity, with significant activities also being seen with kaempferol, apigenin, biochanin A and daidzein. The importance of hydroxyl groups at carbon positions 4', 7 andor 6 was shown here, as al1 compounds determined to have estrogenic activity possessed hydroxyl groups at one or more of these positions

(101). Binding to the ER was confirmed through competition assays with [3~]estradiol.

Le Bail et al used a stably-transfected MCF-7 cell Iine with a luciferase reporter gene and €RE to determine estrogen agonist and antagonist activities. Significant agonist activities were seen for compounds containing 7 andor 4' hydroxyl groups at concentrations as low as 1 PM. Moreover, they determined that phenolic hydroxyl groups in positions 4' and 7 could be considered to be equivalent to the position 3 and

17-hydroxyl groups of estradiol (102). These results were also confirmed using a recombinant yeast strain stably transfected with the human ER gene, with upregulation of this gene being the measured endpoint (103). Finally, resveratrol, a red wine polyphenoI, not a flavonoid, but possessing a similar structure, has also been shown to bind to ER in the ['~l]estradiolcompetition assay, and to induce luciferase production in both MCF-7 and T47-D cell lines transfected with ERE-tkl09-luc or ERE2-tkt09-luc plasmids. Resveratrol also increased progesterone receptor mRNA expression and pS2 expression in MCF-7 cells at 10 pM concentrations (104).

Several researchers have investigated the androgenic and anti-androgenic activities of flavonoids. None have found these compounds to have agonist androgenic activities, but most have observed anti-androgenic (105) and/or inhibition of androgen- mediated activities. Our group evaluated inhibition of prostate-specific antigen (PSA) production in an ER (+), AR (+) breast cancer cell line (BT-474) and prostate cancer cell transfected with the human AR (PC-3(AR)2). 18 of the 72 flavonoids and related compounds tested demonstrated such inhibition of PSA production at concentrations of

10 pM. Several compounds, including genistein and biochanin A, had blocking effects on PSA production at concentrations as low as 0.1 pM (106,107). Davis et al examined modulation of PSA expression in the LNCaP (androgen-dependent) VeCaP (androgen- independent) prostate cancer cell lines by genistein. This isoflavone had differential effects on PSA expression in the two ce11 lines. PSA mRNA and protein expression and secretion were suppressed in the LNCaP cell Iine at al1 concentrations, while only high concentrations of genistein inhibited PSA expression in VeCaP. Inhibition of cell proliferation in VeCaP was independent of PSA signaling pathways, leading the authors to conclude that the anti-proliferative effects of genistein were irrespective of androgen responsiveness in this androgen-refractory cell line (108).

Resveratrol, in concentrations of 50, 100 and 150 pM, was given to LNCaP cells in the presence or absence of 1 nM mibolerone, a non-metabolized, synthetic androgen, to evaluate expression of two androgen-regulated proteins, PSA and human glandular kallikrein 2 (hW), as well as AR. The cells were transfected with either a PSA promoter-luc, hK2 AREIminimal thymidine kinase promoter/CAT or an AR promoter-luc construct. Resveratrol treatment completely abolished androgen-induction of the PSA promoter, hence inhibiting production of any PSA. Transfection with triple ARE constructs demonstrated similar results. Western blotting of AR protein levels indicated a dose- and time-dependent inhibition of AR with resveratrol, and ARA70, a CO- activator, was found through Northern blotting to decrease, with a maximum reduction using 100 pM resveratrol. These results indicate that AR-mediated gene expression and cell processes are affected by resveratrol, and, moreover, repression of expression of ARA70 may further enhance inhibitory effects of this red wine polyphenol on androgen action (109). Green tea polyphenols have been shown to have similar repressive effects on androgen action in mice (1 10).

Several enzymes responsible for steroid hormone production and metabolism can be inhibited by flavonoids. These include aromatase, sulfatases, hydroxylase enzymes, Sa-reductase, and 17f3-hydroxysteroidoxidoreductase enzymes (Figure 1.4).

Aromatase is a cytochrome P450 complex responsible for conversion of androgen to estrogen. Inhibition of this enzyme may thus reduce estrogen-dependent cancers.

Jeong et al tested 28 flavonoids against partially purified aromatase prepared from human placenta. Over 50% of the flavonoids tested significantly inhibited aromatase activii, with greatest activity seen with apigenin, chrysin and hesperetin (111). In preadipocytes, the flavonoid chrysin has been shown to have similar potency and efFectiveness against aromatase as aminogluethimide, a pharmaceutical aromatase DEA-S Steroid sulfatase 1 DHEA 38-MDi 1 4-Dione Aromatase

El Steroid sulfa tase t

Figure 1.4. Main Biosynthetic and lnactivating Pathways of Estrogens and Androgens. DHEA - dehydroepiandrosterone; DHEA-S - DHEA-sulfate; El - estrone; E2 - estradiol; DHT - dihydrotestosterone; Test - testosterone. inhibitor (112). Other flavonoids with anti-aromatase activity include luteolin and kaempferol (1 13). Using radioactive Iabeling and HPLC, various flavonoids were shown to have inhibitory effects on both aromatase and 17fbhydroxysteroid dehydrogenase. A hydroxyl group at C-7 was essential for anti-l7b-hydroxysteroid dehydrogenase activity.

Flavonoids with 7-methoxy or 8-hydroxyl groups on the A ring showed only aromatase activity (1 14).

Inhibition of estrone sulfatase activity, a route for estrogen synthesis, is another mechanism for breast cancer prevention. Quercetin, kaempferol, and naringenin were found to inhibit this enzyme at concentrations of 10 - 150 pM in human liver microsomes (115). These compounds were found to dose-dependently inhibit estradiol metabolism in concentrations of 10 to 500 FM. No influence on estrone formation was observed, while these compounds demonstrated potent inhibition of estriol formation.

The proposed mechanism for these results was described to be inhibition of the cytochrome P450111A4 enzyme, which reversibly hydroxylates 17$-estradiol + estrone

+ estriol (1 16). Similarly, 17fMydroxysteroid oxidoreductase enzymes (178-HSORs) catalyze the reversible reaction between weak estrone (oxidized form) and highly active estradiol. One study (1 17) examined whether flavonoids inhibit reduction of estrone to

17p-estradiol by one of these enzymes, 17B-hydroxysteroid dehydrogenase (17p-MD).

This enzyme is expressed in steroidogenic cells including ovarian granulose cells and target tissues of estrogen action, including normal and malignant breast and endometrium, and in the epitheiium of prostatic urethra and collecting ducts (1 18421).

By measuring conversion [%]estrone and [3~]estradiol,genistein and coumestrol were found to dose-dependently block 17B-HSD. This inhibition was NADPH-dependent. Similarly, in P. testosteronii, this enzyme was competitively inhibited by daidzein and genistein (122). Sa-reductase has also been inhibited by isoflavones in a Sprague

Dawley (SD) rat model (123). These results indicate that flavonoids may exert some of their biological effects by modulating the activities of enzymes responsible for metabolizing steroids critical to hormonal functions. By doing sol dietary flavonoids may help prevent steroid hormone-dependent cancers.

Cell Growth and Signal Transduction

Many studies have investigated proliferative effects of flavonoids, and mechanisms by which stimulation or inhibition of cell growth may occur. Several experiments have examined the biphasic effects of genistein and other isoflavones, which in ER(+) MCF-7 and T47-D cells stimulate cell growth at concentrations of 10 nM

- 10 pM, and inhibit proliferation at concentrations of 50 - 100 pM

(96,98,102,113,124,125).

Over 30 flavonoids have been tested for effects on cell proliferation and potential cytotoxicity, in a variety of cell Iines, inciuding colon cancer cell lines Caco-2 and HT-29, and the breast cancer cell iine MCF-7, for their influence on proliferation and apoptosis, measured through caspase-3. ECsovalues ranged between 40 and 200 PM. In almost al1 cases, inhibition by flavonoids occurred in the absence of cytotoxicity. Only baicalein, myricetin and flavone were able to induce apoptosis in Caco-2 and HT-29 cells. No alterations of cell growth have been seen for taxifolin, hesperitin or catechin in the HT-29 cell line (126,127). in the breast cancer cell Iines tested, 7-hydroxyflavone,

7,8-dihydroxyflavone and kaempferol did not alter proliferation either (1 02). Other flavonoids that are inhibitory at high concentrations in both ER (+) and ER

(-) breast cancer cell lines include quercetin, luteolin, biochanin A, coumestrol, daidzein, kaempferol and apigenin (99,124,120). None were shawn to work through cytotoxic mechanisms. However, when MCF-7 cells were exposed to 50 or 100 pM genistein continuously for up to 1O days, DNA synthesis was strongly inhibited from Day 1-4, after which time cytotoxicity occurred (124). In T47-0 breast cancer cells, 20 pM genistein, quercetin and kaempferol markedly inhibited growih. Only quercetin was inhibitory over the entire concentration range of 100 nM - 20 PM. At the highest concentrations, extensive chromatin fragmentation was observed, suggesting apoptosis (96). This is similar to short-term effects of flavone, which has been observed, through semi- quantitative RT-?CR, to inhibit cell proliferation through apoptosis. Flavone was highly selective towards transfomed cells only, and thus may be important for chemotherapeutic uses in colon cancer (127).

No obvious structure-function relationships have been observed for effects on proliferation, either for type of flavonoid (isoflavone, flavone, fiavanone) nor hydroxylation pattern (102,126).

At concentrations greater than 25 PM, the soy isoflavones genistein, biochanin A, equol, and to some extent daidzein have been shown to modulate the proliferative activity of environmental carcinogens, including O$-DDT, 4-nonylphenol and 5- octylphenol. Genistein was the must potent, with an IC50 of 25 - 33 FM (129).

Therefore, diet-derived flavonoids may be protective against environmental xenoestrogens. Mechanisms for inhibition of proliferation involve signal transduction pathways, including effects on protein kinases, epithelial growth factor (EGF) and vitamin D receptors, insulin-like growth factor (IGF-1) and transforming growth factor (TGF-g)

(Figure 1S).

Kinases are enzymes involved in signal transduction, responsible for conversion of 1-phosphatidylinositoI to inositol 1,4,dtriphosphate (1P3). Increased levels of IPs have been found in clinical samples human carcinomas and tissue culture cell lines, including cancers of breast and ovary, compared with normal cells (130). Of I4 flavonoids evaluated in one study, myricetin was found to be the most potent inhibitor of

IP3-kinase activity, with an lCso of 1.8 pM. Apigenin, fisetin and quercetin were also effective. Structure-function analysis of flavonoid-induced inhibition of IPykinase, indicated the importance of position, number and substitution of hydroxyl groups on the

B-ring, and saturation of the C-2 - C-3 bond (131). Weber et al found that quercetin and genistein down-regulated Ips in both a time- and dose-dependent fashion. This led to marked reduction of IP3concentration, then to apoptosis (132).

Tyrosine kinases are ubiquitous enzymes involved in many processes, including those responsible for growth and differentiation. Several studies have investigated the effects of flavonoids on this group of enzymes. Genistein has been found to be the strongest inhibitor of tyrosine kinases in several cell lines (133,134). Apigenin and kaempferol have also been demonstrated to dose-dependently inhibit both EGF receptor kinase autophosphorylation and mitogen activated protein (MAP) kinase

(131 ,I35), and to decrease phosphotyrosine content (136). However, other studies 1 Ligand

Plasma membrane

Protein tyrosine kinases

BIOLOGICAL RESPONSE

Figure 1.S. Signal Transduction Pathways influenced by Flavonoids. IP3- inositol triphosphate. Pathways found to be influenced by flavonoids are in bold, blue pnnt. have conflicting results, in both human breast cancer (137), and prostate cancer ceil lines (138). These studies concluded that a more distal event in the EGF receptor- mediated signal transduction cascade was involved for cell growth inhibition.

Experiments with the tea catechins, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) demonstrated that low concentrations these compounds may activate MAP kinase, leading to the expression of survivai genes c-fos and c-jun. At suprapharmacological concentrations, these fiavonoids induce non-specific necrotic cell death (139). Genistein, on the other hand, has been found to down-regulate EGF- induced c-fos transcription in MOA-ME-468 human breast cancer cells (134). Various flavonoids and metabolites have also been shown to modulate cytochrome P450s responsible for site-specific 7a-,68- and 2a-hydroxylation of testosterone (140).

Inhibition of cell proliferation by genistein and other flavonoids, have been shown to be associated with specific arrest of the cell cycle. Genistein treatment of MCF-7 cells (10 PM) causes reversible arrest at the G2/M phase of the cell cycle (141).

Quercetin arrests the cell cycle at G1 and S-phase boundary (Table 1.3). Moreover, the two flavonoids together have been shown to synergistically inhibit growth in OVCAR-5 ovarian cancer cells (142). One mechanism of cell cycle arrest and apoptosis by apigenin, luteolin and quercetin has been shown to be through wild type p53 accumulation (143). Synergism has observed in cell growth inhibition, cytotoxicity and reduction of IP3 concentrations with tarnoxifen and genistein treatment of MDA cells

(144). These results may be of interest in clinical treatment of breast and ovarian cancers. Table 1.3. Cell Cycle

Phase Function G1 (and GO) Gene expression and protein synthesis. Regulated primarily by extemal stimuli. Allows ceIl to grow and produce proteins needed for next phase. CeIl may enter GO instead and remain 1 quiescent.

(M 1 Mitosis - ceIl divides into two daughter cells.

Other mechanisms invoived may include inhibition of other nuclear receptors,

such as the vitamin D and retinoic acid (RXR-a) receptors. Downregulation of these

two receptors has been shown with treatment of human keratinocytes with apigenin,

quercetin and fisetin (145). Finally, induction of p21WAFEIPI expression may also be important. A 3 - 4-fold induction of this protein has been shown to precede apoptosis

(125, 145-149).

Antioxidant Activities

Flavonoids have been shown to have both antioxidant and pro-oxidant activities

in vitro, and in animal models. Activities and structural requirements for these activities

have been deterrnined by several investigators. However, conflicting potencies have

been reported, dependent on the assay and methods used.

Using an oxygen radical absorbance capacity assay, and three different reactive

species - a peroxyl radical generator, a hydroxyi radical generator, and CU'+, a

transition metal - Cao et al determined both antioxidant and pro-oxidant activities of various flavonoids, and structure-function relationships. They found that both

antioxidant and Cu2* pro-oxidant activities were dependent on the number of hydroxyl

substitutions, with compounds that contained multiple hydroxyl groups showing anti-

peroxyl radical activities several times stronger than Trolox, an a-tocopherol analogue.

Dihydroxyl substitutions at C-3' and 4' were particularly important to peroxyl radical

absorbing activity. O-methylation of hydroxyl groups inactivated both antioxidant and

pro-oxidant activities (150). Flavonoids including naringenin, naringin, hesperetin and

apigenin were also found to form pro-oxidant metabolites that oxidized NADH and

glutathione upon oxidation by peroxidaselhydrogen peroxide. The implications of pro-

oxidant activities of flavonoids is uncertain (151,152).

The importance of the 4' hydroxyl group and 5',7 dihydroxy was deterrnined

through examination of antioxidant activity in the aqueous phase through ABTS+ total

antioxidant activity assay (Figure 1.1). The order of reactivity for isoflavones in radical

scavenging was genistein>daidzein, genistin, biochanin A, daidzin. No activity was

seen for formononetin or ononin (153). Examining other flavonoids, quercetin,

cyanidine, which contain 3',4'-dihydroxy substituents and conjugation between A and 8

rings have antioxidant activities four-fold greater than that of Trolox. Kaempferol, which

does not have the ortho-dihydroxy substitution, and catechin and epicatechin, which do

not possess the 2,3 double bond, have greater than 50% reduction of antioxidant

activity. Free radical scavenging was highest with EGCG, then quercetin (50% less

effective). Genistein, daidzein, hesperetin and naringenin were not effective (154).

Hydrogen donating ability of a range of phytoestrogens were assessed using

electron spin resonance spectroscopy, the ferric-reducing ability of plasma assay and Trofox equivalent antioxidant capacity. The ability of compounds b inhibit lipid peroxidation was also examined in vitamin E-deficient liver microsomes. Only kaempferol was found to have significant antioxidant activity in this study. Genistein had the highest activity of the isoflavones, however, isoflavones as a group were relatively poor hydrogen donors. Equol and coumestrol had higher potencies than isoflavones, but they too were weak (155,156). Inhibition of microsomal lipid peroxidation by isoflavones and metabolites were found to have the relative activities of isoflavan > isoflavanone > isoflavone (1 57). In 12-0-tetradecanoyl phorbol 13-acetate

(TPA)-activated HL-60 cells, genistein had potent activity against hydrogen peroxide.

Daidzein had moderate antioxidant effects, and apigenin and biochanin A had no effect.

In contrast, genistein, apigenin and prunectin were equally potent in inhibiting superoxide anion generation by xanthinelxanthine oxidase. For both systems, hydroxyl substitution at 4' position was important (158).

Finally, rats gavage-treated with tangeritin, chrysin, apigenin, naringenin, genistein and quercetin were analyzed for effects on phase I and II enzymes in liver, colon and heart, and antioxidant enzymes in red blood cells. Glutathione-S-transferase was significantly induced by apigenin, genistein and tangeritin in heart, but not colon or liver. In red blood cells, chrysin, quercetin and genistein significantly decreased activities for glutathione reductase, catalase and glutathione peroxidase. Superoxide dismutase was only inhibited by genistein. Moreover, chrysin, quercetin, genistein and

0-naphthoflavone were significantly protective against 2-amino-1-methyl-6- phenylimidazo[4,Bb]pyridine (PhlP)-DNA adduct formation in the colon, suggesting a feedback mechanism (159). Black tea also strongly reduced PhlP-DNA adduct formation (67%) in the colons of male Fischer (F) 344 rats (160). However, for many of

these in vitro and animal studies, high concentrations of flavonoids were used. Several

researchers have therefore questioned the physiological relevance of these antioxidant

effects (161,162).

Carcinoaenesis

Flavonoids have been demonstrated to reduce carcinogenesis in animal models

and in vitro, and to modulate enzymes implicated in the carcinogenic process. Their

effects on initiation and promotion stages of the carcinogenic process have been

evaluated, and several mechanisms have been proposed, including influences on

development and hormonal activities.

The initial animal work was performed by Troll et al, who demonstrated a

significant reduction of mammary cancer by X-irradiation in SD rats fed a raw soybean

diet, compared to those fed casein (44% developed tumors versus 74%). Although this

protection was attnbuted to protease inhibitors present in soy, other compounds,

including isoflavones could have been at least partially responsible (163). Barnes and

Messina have conducted many animal studies using soy and isoflavones in chemical

carcinogenesis models of mammary and other tumors. The majority of these show

protective effects (6).

The soy isoflavones genistein, daidzein and biochanin A, as well as soy protein

and soy milk have al1 been used to determine effects on mamrnary carcinogenesis.

When female SD rats were fed a high fat basal diet containing 10% fermented soy milk

or 0.02% or 0.04% isofiavone mixture during and after PhlP exposure, mammary tumors were significantly smaller, and tumor multiplicity significantly lower than controls

(164). Simiiarly, when SD rats were fed a 10% soybean or 10% miso supplemented diet, or were given 10 or 50 mglkg body weight biochanin A for 18 weeks, tumor multiplicity was significantly decreased. The 50 mglkg biochanin A dose als~ significantly reduced tumor incidence compared to control (32% versus %O%), and proliferation cell nuclear antigen labeling index. When MCF-7 cells or MDA-ME-468 cells were treated with 15, 30 or 45 pM genistein, then implanted into a nude mouse xenograft rnodel, the tumorigenic potential of the cells was diminished (165). Not ail studies agree with these results. SD rats fed one of 4 purified diets containing casein or soy with 0.03, 0.4 or 0.81 mg isoflavones for 2 weeks, then treated with 7,12- dimethylbenz[a]anthracene (DMBA) or peanut oil, showed only a trend (p<0.09) for an inverse relationship between tumor incidence and isoflavone intake (1 66). Cohen et al found no significant inhibition of nitrosomethylurea- (NMU) induced mammary carcinogenesis for rats taking soy protein-supplemented diets (167).

Timing of isoflavone treatment may be important. SD rats were exposed to 0, 25 or 250 mg genisteidkg AIN-76A diet from conception to Day 21 post-partum. At Day 50 post-partum, al1 were treated with 80 mg DMBAlkg body weight to induce mammary cancer. Once sacrificed, it was found that genistein feeding resulted in a dose- dependent protection against the development of mammary tumors, with fewer tumors per rat. Moreover, it was found that the mice given genistein had fewer terminal end buds (TEBs), which are mammary structures most susceptible to carcinogenesis.

(168). These results were confinned in a similar study (169). 500 pg/g body weight genistein given subcutaneously to prepubertal female SD rats prior to DMBA administration at Day 50 also resulted in fewer TE& and more lobules (170). It was concluded that genistein could suppress the development of chemically-induced mammary cancer without reproductive or endocrinological toxicities (171).

Genistein, daidzein, their glycosylated forms (genistin and daidzin), as well as soy products have also be protective against early stages of 3,2'-dimethyl-4- aminobiphenyl (DMA6)-induced prostate cancer (172), and azoxymethane (A0M)- induced colon cancer in rat models (173). Therefore, although not al1 studies show chemopreventive effects, the vast majority indicates that soy isoflavones and food sources may be useful in preventing steroid hormone-dependent cancer development.

Other flavonoids evaluated for anticarcinogenic effects in the colon include quercetin, rutin and hesperidin. Female CF1 mice were treated with 0.1, 0.5 or

2.0°h/diet quercetin, or 1.O or 4.0%/diet rutin for 50 weeks to assess inhibition of AOM- induced colonic neoplasia. No alterations were seen for mice fed flavonoid- supplemented diets without AOM treatment. However, among AOM-treated mice, both

2.0% quercetin and 4.0% rutin significantly inhibited hyperproliferation and the shift of S- phase cells to middle and upper portions of the crypts. Moreover, significantly fewer focal areas of dysplasia (FADs) were found with both of these treatments, and 2.0% quercetin reduced tumor incidence. 4.0% rutin showed a trend for lower tumor incidence (174). A second study performed by this group fed quercetin supplementation of 0.5, 2.0 or 5.0%, or 2.0% or 4.0% rutin with 5% or 20% corn oil to female CF1 mice for 9 weeks. 80% of quercetin-fed mice on the high fat diet remained free of FADs, compared to 29% of those on the high fat control diet (pe0.01). For rutin, the difference was bordering significance (p<0.08) (175). Similar results for AOM- induced mice have been shown by other groups (176). Quercetin- and rutin- supplementation in normal mice have also been shown to increase numbers of colonic epithelial cells per crypt column, apoptotic index and redistribution of apoptotic cells along the crypt axis. Quercetin alone has also been observed to induce FAD in normal mice fed AIN-76A diets (1 75).

Hesperidin, the major flavanone in orange juice has been shown to inhibit chemically induced colon carcinogenesis (177), and double strength orange juice slows down DMBA-inducsd mammary cancer in rats (1 78). 8ased on these studies, Miyagi et al tested the hypothesis that not-from concentrate orange juice given to male F344 rats instead of drinking water could inhibit AOM-induced colon cancer. Cancer was initiated by 2 subsequent injections of AOM at 22 and 29 days of age, and on Day 35, half of the rats were put on orange juice + modified diet to equilibrate the macronutrient profiles between the two groups. At 33 weeks of age, the rats were sacrificed and colons analyzed. A 22% reduction in tumor incidence was found for the group fed orange juice compared to control (p<0.05), with a trend towards srnaller average tumor burden

(p=0.13). The labeling index and proliferation zone were lower, and enhanced cell differentiation was found in the bottom two-thirds of the crypt (179). Pure hesperidin and diosmin, also found in citrus fruits, were fed for 5 weeks (initiation treatment) or 28 weeks (post-initiation treatment) at levels of 1000 ppm each or in combination (900 +

100 ppm) to AOM-induced male F344 rats. At the end of 32 weeks, incidence and multiplicity of neoplasms with or followed by flavonoid treatment were significantly smaller than with AOM alone (p<0.001). The combination of the two flavonoids was not better than each alone. 00th significantly inhibited development of aberrant crypt foci, and were significantly associated with lower labeling index and other markers of carcinogenesis (177). These studies indicate that flavonoids, either purified or within foods, are chemopreventive against colon carcinogenesis.

Cyclooxygenase 2 (COX-2) and cytochrome P450s are enzymes involved in the

carcinogenic process that can be modulated by flavonoids. Of 12 flavonoids tested in

the DLD-1 human colon cancer cell Iine, quercetin was the most potent suppressor of

COX-2 transcription, with an lCso of 10.5 PM. Catechin and epicatechin also

demonstrated weak inhibition of COX-2. Structure requirements for inhibition were

found to be hydroxyl groups on the B ring and an 0x0 group at the 4-position of the C

ring (180). In lipopolysaccharide-activated macrophages, apigenin, genistein and

kaempferol al1 markedly inhibited transcriptional activation of this enzyme with lCsosless

than 15 PM, with apigenin being most potent (181). These observations are important

because COX-2 is linked to the loss of growth control and apoptosis in various tumors

(182). In colorectal tumors, over-expression of this enzyme occurs in almost 90% of

cases (183), hence it is a key target of pharmacotherapy (184). In HT-29 cells exposed

to 150 pM flavone for 48 hours, COX-2 mRNA bels were strongly reduced.

Transcription of NF-KB, a transcription factor able to inhibit apoptosis in cancer cells

was also inhibited by flavone. Inhibition of both of these proteins may help to reduce

chemoresistance of cancer cells (127).

Finally, several cytochrome Pa0 enzymes induce carcinogenesis through

metabolism of procarcinogens to ultimate carcinogens. Several studies have assessed

the ability of various fiavonoids to inhibit activation of benzo(a)pyrene (BaP) or 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD). Analysis of 23 flavonoids found that flavonols or flavones with free 5- or 7-hydroxyl groups are potent inhibitors of P450-mediated metabolism of BaP in Iiver S9 homogenate from rats (185). Genistein, daidzein, genistin and daidzin were found to block TCDD-induced CYP1A1 activation. This inhibition was correlated with the capacity of these isoflavones to prevent CYP1A1- mediated covalent binding of BaP metabolites to DNA (186). Inhibition of these enzymes may thus represent one mechanism by which flavonoids are protective against carcinogenesis.

1.2.1 -4 Influences on Modulators of Risk for Steroid Hormone-Dependent Cancers

Few clinical trials have been conducted to determine the influences of flavonoids on risk factors for steroid hormone-dependent cancers. Most of those that have been conducted, have looked at potential modulation of breast cancer risk factors by soy isoflavones. The results of these studies are conflicting, and confusing.

Urinary excretion of isoflavones have been assessed in many studies as a marker of isoflavone consumption. To detemine whether this method is reliable, several groups have correlated database calculations for isoflavones based on self- reporting and urinary isoflavone excretion. All have shown very significant correlations, aIong a broad range of intakes (187-190). Therefore the use of food frequency questionnaires for rneasurernents of soy intake appear to be relatively reliable.

Risk factors that have been associated with decrease in risk of breast cancer include lower plasma concentrations of ovarian hormones, higher levels of sex hormone binding-globulin (SHBG), longer menstrual cycle, particularly the folticutar phase, and high urinary isoflavone excretion. Therefore, many of the clinical triais conducted wers to determine the effects of soy feeding on these endpoints. In one of the initial soy feeding studies, Cassidy et al examined the effects of soy feeding on ovarian cycle. 60 g of soy protein, providing 45 mg isoflavoneslday significantly (p<0.01) increased follicular phase length andlor delayed menstruation. Moreover, midcycle surges of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly suppressed, and plasma estradiol concentrations in the follicular phase increased.

These results were exciting, as similar responses have been seen with tamoxifen, when used as a prophylactic agent against breast cancer (191).

Since then, many other clinical trials have been conducted, Lu et al have examined the modulation of soy feeding on these parameters in several studies. In one study, 10 healthy, cycling premenopausal women were given 36 ounces of soy milk

(equivalent to 113-207 mg isoflavones/day) for one month. A 25% reduction in estradiol, and a 45% reduction in progesterone, were found, both highly significant

(p~0.01, pc0.0001, respectively). No changes in LH or FSH were found (1 92).

However, in a randomized cross-over study, using soy protein powders with low isoflavone (64 mglday), high isoflavone (128 mglday) or control, for 3 cycles plus 9 days, the low isoflavone diet significantly decreased LH (p=0.003) and FSH (p=û.04), during the periovulatory phase. The high isoflavone diet also reduced free thyroxine and dehydroepiandrosterone sulfate (DHEAS) during early follicular phase. Besides estrone, which decreased during mid-follicular phase, no other significant changes in hormones or length of cycle were found (193). Several other feeding studies have shown similar canflicting results (194-196). Length of treatment and levels of soy do not seem to influence results. Higher levels of sex hormone binding globulin have been associated with decreased breast cancer risk. This plasma protein binds to steroid hormones

(estrogens and androgens), thereby "inactivating" them, i.e. only free steroids exert their

effects. Therefore, SHBG is one mechanism through which steroidal activity can be

rnodulated (197). Significant positive correlation between urinary excretion of

phytoestrogens and p!asma SHBG, and negative correlation between SHBG and

excretion of 1Ga-hydroxyestrone (see below) and estriol have been found (198). These

results suggested that isoflavones may affect uptake of sex hormones by regulation of

plasma SHBG levels and influence biological activity. However, other studies, do not

support this hypothesis (191,199).

Higher levels of two putative carcinogenic metabolites of estradiol hzve also

been implicated in breast cancer risk. These are 4-hydroxyestrogen and 16a-

hydroxyestrogen. Lower amounts of the anticarcinogenic metabolite 2-hydroxyestrogen

are also associated with greater breast cancer risk. In order to determine whether a soy

diet may alter the metabolism of 17$-estradiol to these products, eight women were

placed on the soy milk-supplemented diet described above for one complete rnenstrual

cycle. Aiter a four-month washout, they were then given an isoflavone-free soy milk

supplement, providing less than 4.5 mg isoflavones/day. It was found that the

isoflavone diet increased mean daily urinary excretion of 2-hydroxyestrone by 47%

(p=0.03), but had no effect on 16a-hydroxyestrone production. Because the ratio of

2:16a also significantly increased, it was suggested that isoflavones, through increased

metabolism of endogenous estrogens to the protective 2-hydroxy fon, may be

important in lowering 17p-estradiol levels (200). The role of ethnicity has been a consideration for breast cancer development, as

Asian women have significantly lower incidence. Hence, Wu et al placed 20 premenopausal women, 10 Asian-American, 10 non-Asian American, on a 7-month soy intervention study. Asian soy foods, including tofu, soy milk and green soybean peas, providing 32 mg isoflavones/day, were added to a basal diet for 3 menstrual cycles.

The intervention was found to significantly reduce serum estradiol in the luteal phase in the Asian women only. Moreover, these women had higher excretion levels of isoflavones, compared to their non-Asian counterparts. It was concluded that perhaps genetics does play a role in the metabolism of these compounds, and thus modulation of breast cancer risk factors (201). Opposing this theory, is a study of 31 premenopausal Japanese women, fed 400 mL soymilk per day, providing 109 mg isoflavones, for 3 consecutive menstrual cycles. Although estrone and estradiol levels decreased 23% and 27%, respectively in the soymilk group, these were not statistically significant (202). Perhaps a longer study would have enabled these results to reach significance.

Another parameter that may be important in determining breast cancer risk is the profile of breast secretions. Petrakis et al hypothesized that long-term consumption of commercial soy protein products would alter features of nipple aspirate fluid (NAF) of non-Asian women to resemble those previously characterized in Asian women. 24 normal pre- and postmenopausal Caucasian women underwent this year-long study.

For months 1-3 and 10-12, they did not eat any soy products. Between months 4 and 9, these women consumed 38 g of soy protein isolate, (SPI) containing 38 mg of genistein.

NAF volume, gross cystic disease fluid protein (GCDFP-15) concentration and NAÇ cytology were used as biomarkers of possible effects of soy protein isolate on breast.

Other measurements taken were plasma concentrations of estradiol, progesterone,

SHBG, prolactin, total chofesteroi, HDL-C and triglycerides. Cornpliance was measured through urinary excretion of genistein and daidzein, and was excellent throughout the study (203).

Compared to months 1-3, there was a 2-6-fold increase in NAF volume in months

4-9 for al1 premenopausal women, and no change in postrnen~pausalsubjects. No changes were seen in any of the plasma parameters for postmenopausal women.

However, in the premenopausal group, plasma estradid levels erratically increased during soy consumption, and there was a moûerate decrease in GCDFP-15 levels.

What was surprising to investigators, and of potentiai concern, was the cytological detection of epithelial hyperplasia found in 7 of 24 women (29.2%) during months of SPI consurnption. These results indicate that prolongeci consumption of SPI has stimulatory effects on premenopausal breast, suggestive of an estrogenic stimuhs (203). These data were further supported by a randornized trial examining the effects of 60 g soy supplement on the proliferation rates of premenopausal, histologically normal breast epitheliurn of 48 women with benign or malignant breast disease. One group was given a nomal diet, and the other given diet + soy supplement for 14 days. Biopsy sampies of normal breast were labeled with [3~-thymidineto detect the number of cells in S-phase, and were measured for the proliferation antigen Ki67. The proliferation rate of the breast lobular epithelium significantly increased after the 14-day soy supplementation, when bath day of cycle and age of patient were accounted for. PR levels also increased (204). lncreases in pS2 concentrations in NAF have also been found (205). These studies, taken together, demonstrate that both short-term and long- term soy feeding may provide a stimulus for proliferation in the breast. The implications of this remain unknown.

Very few studies have looked at men. We examined the effects of feeding 33 glday soy, providing 86 mg isoflavones/2000 kcaVday, to 31 men and postmenopausal women. Ex vivo hormone activity showed no alterations in androgen (through PSA) or estrogen (pS2) activities (206). Many more studies must be done before we can decipher the data thus far, let alone determine the effects of other flavonoids in steroid hormone-dependent cancers.

1.2.1.4 Discussion

Many mechanisms have been proposed for how flavonoids may help prevent steroid hormone-dependent cancers and several have been validated in vitro and in animal models. These include modulation of steroid hormones, inhibition of proliferation, and anticarcinogenic and antioxidative activities. However, the physiological implications are questioned by most. For most animal models, doses tested are significantly higher than fhose that would exist in human plasma after consumption of whole foods. Nutraceutical or functional food administration has thus been suggested as possibilities for reaching such concentrations.

Randomized clinical trials are stilI in their infancy in this area. To date, only soy has been tested in this capacity, dealing specifically with breast cancer risk factors.

Though several studies have been done, these are very conflicting. Little data exist on other flavonoids or other cancer types. At this point in time, we are still in the dark. We cannot be sure that consumption of soy will have positive, neutral or even negative effects of breast cancer risk. Advising women at high risk for breast cancer, or those who have this disease and want alternate therapies is not feasibie at present.

1.2.2 Steroid Hormone Receptors

Steroid hormone receptors, including ER, AR and PR, are members of the

Nuclear Hormone Receptor Superfamily, which is made up the classic steroid recôptors, thyroid receptors, vitamin D receptor and ietinoic acid receptors - RAR and RXR. All of the members of this superfamily share commun functional domains, lettered A to F,

Starting from the N-terminus, these are the trans-activation, DNA binding, hinge and ligand binding domains (207,208 and references within).

AB: Trans-activatr'on Domain

This domain is weakly conserved among members of the superfamily, and is of variable length. It contains the autonomous activation function (AF) AF-1, and is involved with binding of specific and general transcription factors necessary for transcription of the nuclear receptor-regulated genes. C: DNA-binding Domain

This domain, towards the carboxy terminus of the trans-activation domain, consists of 66-68 amino acids, and is the most highly conserved region among the steroid receptors. It contains 20 invariant amino acids, forming two zinc finger DNA binding motifs, which provides the basis for protein-DNA interactions with regulator sequences of target genes (209-215). Specifically, nine of these conserved arnino acids are cysteine residues, of which 8 interact in a coordinated fashion to form two separate tetrahedral metal-binding complexes, or fingers. Four cysteine residues in each finger binds one zn2' molecule, which permits interaction with DNA. The functional role of the second finger is not entirely clear, but studies suggest it aids in the stability of binding of the receptor to DNA (214).

Amino acids adjacent to these cysteines are also important, as they define the specificity of a given receptor binding to its respective response element. It has been shown through mutation studies of the ER, that substitution of glutamine, glycine and alanine, found in ER, by glycine serine and valine, contained by the glucocorticoid receptor (GR), results in a receptor that no longer recognized the ERE, but instead, the glucocorticoid response element (GRE). Therefore alterations of these three amino acids can alter or destroy receptor function.

0: Hinge Domain

This region is involved in homodimer formation, necessary for activation of nuclear receptor. It contains sequences responsible for nuclear localization of steroid receptors, with studies indicating that signais for localization of the receptor into the

~ucleuscan occur even in the absence of ligand binding (210-212).

E: Ligand-binding Domain

This multifunctional, carboxy-terminal half of the protein encompasses the ligand- binding domain, a second activation function, AF-2, a dimerization domain and a region invoived in nuclear localization. The AF-2 autonomous activation domain is composed of an amphipathic a-helix that is highly conserved among nuclear receptors and is critical for transcription activation (216-21 9).

F: Unknown Domain

This final domain is absent in some receptors, including PR, RAR and RXR. It is quite variable and its function is yet unknown.

1.2.3 Steroid Hormone Receotor Sicrnaling

In the absence of steroid ligand, a steroid hormone receptor resides as part of a larger molecular complex with heat shock proteins (HSPs) (220). Once bound by its respective ligand, the receptor becomes phosphorylated at several serine and tyrosine residues (221). Specific conformational changes are subsequently induced in the receptor, resulting in the dissociation of HSPs, receptor dimerization and localization of the receptor into the nucleus. The receptor-ligand complex then binds with a HRE within the promoters of target genes (222). Transcription factors then act to transcribe the gene to mRNA, which then leaves the nucleus and enters the endoplasmic reticulum for translation into protein. The protein then performs its specific physiclogical function (See Figure 1.3 in Section 1.2.2) (84).

The AF-2 site rnay alternately be regulated by coactivators andior corepressors

(223,224) in a ligand-dependent manner. This mechanism supports the theory of usquelchingn,the transcriptional interference between steroid hormone receptors due to lirniting common transcriptional cofactors (223-225). These mediators or coactivators are required to achieve efficient transcription (226-228). Details of these cofactors and mechanisms by which they act are beyond the scope of this project.

1.3 Hypothesis

Flavonoids may have steroid hormone activities, estrogenic, progestational andior androgenic, and thereby may influence the regulation of steroid-hormone dependent genes. Other factors, including the vehicle in which they are found, i.e. natural or processed extract or food, may alter these activities.

1.4 Objectives

1. To develop the techniques and methodology necessary to determine steroid

hormone activity.

2. To assess the agonist and antagonist activities of flavonoids and related compounds

in vitro, and to relate their activity, where possible, to structural properties. 3. To evaluate the steroid hormone activities of several natural products and

commercially prepared nutraceuticals being taken by the North American public,

many of which are sold for hormonal purposes.

4. To determine the steroid hormone effects of one functional food, soy, in healthy

subjects, using Our model system. CHAPTER 2: DEVELOPMEM OF TOOLS TO MEASURE STEROID HORMONE ACTlVlTY CHAPTER 2: DEVELOPMENT OF TOOLS TO MEASURE STEROID HORMONE ACTlVlTY

2.1 Rationale

In order to evaluate the steroid hormone activities of flavonoids, related compounds and metabolites, the tools necessary for making these assessments had to first be developed and tested. These included cell lines, indicator proteins, the assays needed to rneasure these proteins, and the positive and negative controls - namely the steroids and steroid antagonists.

The cell Iine first chosen was the BT-474 human breast cancer cell line, because it was positive for al1 three receptors (estrogen, ER; androgen, AR; and progesterone,

PR). Dr. Pascal Pujol, at the University of Montpellier, Villeneuve, France, determined through a competitive PCR technique (229) that the ER present in these cells was exclusively ERa. This distinction is important because ERP has been shown to have a significantly greater affinity for genistein than €Ra (230). The clinical implications of this difference is yet unknown.

A prostate cancer cell line (PC-3) was then added, which is a receptor-negative cell line that has been stably transfected with the human AR cDNA. This cell Iine was developed by Dr. Theodore Brown, Department of Obstetrics and Gynecology, Mount

Sinai Hospital. The subclones PC-3(A& and PC-3(AR)6 were tested for their upregulation of PSA and other androgen-responsive genes, and used for relevant studies.

The immunoassay for PSA, which is regulated by androgens and progestins, was already developed and tested in the lab, so the requirement was to develop an irnmunoassay to measure pS2, an estrogen-regulated protein. This pS2 assay is a competitr've, enzyme-linked immunosorbent (ELISA)-type assay, and uses a pS2 antibody (Ab), a biotinylated 28-mer pS2 peptide, and a sheep anti-rnouse imrnunoglobulin G (SAMIg) secondary Ab. The pS2 Ab was purchased from

Neomarkers, Union City, CA, and the pS2 peptide was a gift from Dr. Atul Tandon.

After developing this assay, pS2 upregulation was measured after stimulation by estradiol and other steroids. This was necessary in order to qualify pS2 as a good maiker of estrogen activity and regulation.

The final study in this area was to test the effectiveness of clinically-used antagonists to block steroid hormone activity in this system. The effectiveness of ICI

182,780 (faslodex, a pure antiestrogen) to inhibit pS2 production, and the antiprogestin

RU 486 (mifepristone) to inhibit PSA production, was determined in the BT-474 human

breast cancer cell line. The cross-reactivity of the two antagonists was then

determined.

2.2 Development and Evaluation of a Competitive Time-Resolved lmmunofluorometric Assay for the Estrogen-Regulated Protein pS2

2.2.1 Introduction

pS2 is a secreted (231), estrogen-regulated protein (232), classified as a

member of the trefoil family of proteins (233). It is expressed in normal tissue, primarily

in the stomach (234,235), but is also found in normal breast epithelium and othet sites

(236,237). Like human intestinal trefoil factor (hlTF) and human spasmolytic

polypeptide (hSP), the other two family members, the functions of pS2 have not been

completely resolved. However, current research is focussing on its motogenic

capabilities and its role in tissue healing and repair (238-242). pS2 was first discovered in MCF-7 breast cancer cells by virtue of its estrogen- controlled reguiation (232). The presence of this protein in breast tumors has been shown to be a favoutable prognostic indicator (243). It is associated in many studies with estrogen (ER) and progesterone receptors (PR) (243). pS2 has also been used in- vitro to measure estrogenic activity of natural and synthetic compounds (244). To date, pS2 has been measured with Northern blot (245), immunohistochemistry (246), and with immunoradiometric assays (96). To Our knowledge, only one IRMA-type assay is currently commercially available for this interesting protein.

The scarcity of quantitative assays for pS2 protein suitable for anaiysis of tissue culture supematants and other biological sarnples prompted us to develop this method which, we then applied to practical applications. By using the developed assay we here show the mode of regulation of pS2 protein by steroid hormones in the breast carcinoma cell line BT-474.

2.2.2 Materials and Methods

BT-474 and MCF-7 human breast cancer ceIl lines were obtained from the

American Type Culture Collection (ATCC), Rockville, MD, USA. All steroids were from

Sigma Chemical Co., St. Louis, MO, USA. Stock, 10~M solutions of steroids were prepared in absolute ethanol. The secondary antibody (sheep anti-mouse IgG, SAMIg} was purchased from Jackson ImmunoResearch, Westgrove, PA, USA. The pS2 rnouse monoclonal antibody (catalog no. MS-Ill-PABX) was obtained from NeoMarkers, Union

City, CA, USA. pS2 peptide was a gift from Dr. Atul Tandon. The sequence of the peptide, which reprssents 28 amino acids of the carboxyterminus of native pS2 is NHT

KGCCFDDTVRGVWVCWPNTIDVPPEEE.

DifIunisal phosphate (DFP)was synthesized in Our laboratory. The stock solution of DFP was 0.01 moVL in 0.1 moVL NaOH. Alkaline phosphatase-labeled streptavidin

(SA-ALP) was O btained from Jackson IrnmunoResearch as a 1 g/L solution. Working.

SA-ALP solutions were prepared by diluting the stock solution 20,000-foId in a bovine serum albumin (BSA) diluent (described below). White, opaque, 12-well microtiter strips were obtained from Oynatech Labs., Alexandria, VA. The substrate buffer was a Tris buffer (0.1 moUL, pH 9.1) containing 0.1 mol of NaCl and 1 mm01 of Mg& per liter. The substrate working solution (DFP, 1mrnoVL in substrate buffer) was prepared just before use by diluting the DFP stock solution IO-fold in the substrate buffer. The SA-ALP diluent was a 60 g/L solution of BSA in 50 mrnoVL Tris buffer, pH 7.40, cuntaining 0.5 g/L sodium aride. The wash solution was prepared by dissolving 9 g of NaCI, and 0.5 g crf polyoxyethyenesorbitan monolaurate (Tween 20) in 1 L of a 10 mmoUL Tris buffer, pH 7.40. The developing solution contained 1 mol of Tris base, 0.4 mol of NaOH, 2 rnmol of TbCI3, and 3 mmol of EDTA per liter (no pH adjustment).

2.2.2.1 Instrumentation

A time-resolved fiuorometer, the CyberFluor 615 lmmunoanalyzer (MDS Notdion,

Kanata, ON, Canada), was used to measure 7ba fluorescence in white microtiter wells.

The procedure bas been described in detail previously (247-249). 2.2.2.2 pS2 Standard Solutions

MCF-7 breast cancer cells were grown to confluency in 75 rnL flasks with 50 ml of media (phenol-free RPMl with 10% fetal bovine serum, 10 mg/L insulin, 200 rnmoVL

L-glutamine (Gibco BRL, Gaithersburg MD, USA). The flasks were stimulated with IO-' moUL estradiol and incubated for 8 days at 37OC, 5% COn. The supernatants were then harvested and pooled. This stock solution was calibrated as described below.

2.2.2.3 Procedures

Coating of microtiter plates with pS2 mAb. We coated polystyrene microtiter wells by incubating overnight 500 ng/100 pL per well of sheep-anti-mouse immunoglobulin

(SAMIg) in a 50 mmoVL Tris buffer, pH 7.80. The wells were then washed six times with the wash solution and incubated for 2 hours with 100 PL per well of pS2 mAb diluted 1:4,000 (25 ng/100 pl) in 6% BSA. The strips were then washed another six times.

BiotinyIatr'on of pS2 peptide. Biotinylation was performed with sulfosuccinimidyl 6-

(biotinarnido) hexanoate (NHS-LC-LC-Biotin) obtained from Pierce Chernical Co.,

Rockford, IL. Approximately 1 mg of NHS-LC-LC-Biotin was reacted with 200 pg of the peptide in 1 rnL of a 0.5 M carbonate buffer, pH 9.1. After Motinylation for 1 h at room temperature, the peptide was stored at 4°C and used without any further purification.

The concentration of the stock biotinylated peptide solution was 200 pg/mL. pS2 calibrators. MCF-7 supematants were analyzed for pS2 concentration using an irnmunoradiornetric kit (IRMA) (Cis-BIO, Gif-sur-Yvette Cedex, France). pS2 calibrators of 0,62, 125,250, 500 and 1000 ng/mL were prepared by diluting the standard stock in a 50 mmoVL Tris buffer, pH 7.80, containing 60 g of BSA and 0.5 g of sodium azide per liter. pS2 assay. Calibrators or samples (100 pL) were pipetted into coated microtiter weIIs and 50 PL of biotinylated peptide diluted 20,000-fold in 6% BSA was added

(approximately 0.5 ng of peptide per well) . The plates were incubated with mechanical shaking for 1 h at room temperature and then washed six times. To each well we then added 100 pL of SA-ALP conjugate diluted 20,000-fold in the SA-ALP diluent

(approximately 5 ng of conjugate per well), incubated for 15 min as described above, and then washed six times. To each well WEI then added 100 pL of the 1 mmoVL DFP working substrate solution and incubated for 10 min as described above, We added

100 PL of developing solution to each well, mixed by mechanical shaking for 1 min, and measured the fluorescence with the time-resolved fluorometer essentially as described elsewhere (250). The calibration curve and data reduction were performed automatically by the CyberFluor 615 Immunoanalyzer.

2.2.2.4 Tissue Culture Ex~erimentswith Cell Lines

BT-474 breast cancer cells, positive for estrogen, progesterone and androgen receptors were grown to confluency in phenol-free RPMl media supplemented with 10% fetal calf serum, insulin, and L-glutamine at 37"C, 5% COz. They were then transferred to 24-well microtiter plates (Corning no. 25280), where they were grown to confluency in media containing charcoal-stripped fetal calf serum instead of the regular fetal calf serum. They were stimulated with either estradiol, aldosterone, dihydrotestosterone

(DHT), dexamethasone, norgestrel, mibolerone at 1O" M. or alcohol. They were incubated for 7 days, at which time their supernatants were harvested and measured for pS2 concentration.

Time Course Study

The same protocol as above was used, with the exception of the incubation period being 1,3 or 5 days.

Dose- Res~onseStudy

BT-474 cells were grown and plated as above. CelIs were then stimulated with each one of six steroids at to IO-? M, or alcohol (control) and incubated for 7 days.

At this time, supernatants were harvested and analyzed for pS2 concentration.

2.2.3 Results

2.2.3.1 Antibodv Selection and Assav Optirniration

Two mouse monoclonal antibodies and one rabbit polyclonal antibody were initially evaluated to devetop the pS2 immunofluorornetric assay. Among al1 possible assay configurations, as described elsewhere (251 ), we found t hat indirect

immobilization of the primary antibody and use of the monoclonal antibody described above, along with a one-step protocol, gave the best overall results. Other variables,

including incubation times and amounts of reactants, were optimized with a previously

published optimization strategy (251). The final conditions are described in "Methods". 2.2.3.2 Calibration Curve. Detection Limit and Precision

A typical calibration curve of the proposed assay is shown in Figure 2.1. The

detection iimit, defined as the concentration of pS2 detected with a precision of 20% is

16 ng/mL. Within-run and between-run precision was assessed at various pS2 concentrations of tissue culture supernatants behnreen 30 and 300 ng/mL. CVs ranged from 3 to 12% within this range, which are typical for this type of assay.

2.2.3.3 Tissue Culture Ex~eriments

In Figure 2.2 we present data showing conclusively that the pS2 protein

expression is regulated by estrogens only. All other tested steroids had no detectable

effect on pS2 regulation. Furthemore, as shown in Figure 2.3, pS2 secretion and

accumulation into the tissue culture supernatant is time-dependent following stimulation

with estradiol. The effect of estradiol was dose-responsive (Figure 2.4). The effect of

estradiol on pS2 upregulation is evident at estradiol concentrations equal or higher than

IO*" M.

2.2.4 Discussion

We have developed a simple, convenient, and sensitive cornpetitive

immunoassay for quantifying pS2 protein. Unlike Northem blotting and

immunohistochemistry, this methcd allows for secreted or extracted pS2 to be

measured in tissue culture supernatants or in other fluids. The method is efficient since

about 42 samples per plate can be analyzed within 2-3 hours. 50000

J

15000 , O 200 400 600 800 1O00 1200 pS2 Concentration (ng/mL)

Figure 2.1. Typical Calibration Cuwe for the pS2 Competitive Assay Estradiol Aldo Dew Norg Mibo Alcohol Steroids Added

Figure 2.2. pS2 Production by Six Steroids and Alcohol. All steroids were used at a concentration of 10" M. DHT, dihydrotestosterone; Aldo, aldosterone; Dexa, dexamethasone; Norg, norgestrel; Mibo, mibolerone.

2000 r nnn T I

p 1200 (y- 1000 8 800

3 Time in Oays

Figure 2.3. 1ime Course of pS2 Production by Estradiol Estra Norg Mibo Dexa DHT Aldo alcohol nothing

Steroids Added

Figure 2.4. Do,Response of Steroids at Concentrations beîween 1O-' - 10'12 M. For abbreviations see Figure 2. pS2 has been demonstrated in previous studies, and in this one to be an excellent marker of estrogenic activity. Estradiol significantly upregulated pS2 production among al1 of the steroids tested. pS2 production was shown to be suppressed by dexamethasone in other studies (252), as measured by Northern blot.

However, this was not seen in the present study. Furthemore, pS2 expression was shown here to be both time- and estradiol dose-dependent.

A number of clinical applications have already been realized for pS2. This protein is associateci with ER and PR presence in breast tumors (243). It has been shown that pS2 is a predictor of response to tamoxifen treatment (253). Patients with pS2-positive breast tumors show greater relapse-free (243), progression-free and overall survival rates (254,255). In patients with intermediate levels of ER and PR, pS2 has been able to predict tamoxifen effectiveness (256), allowing for therapy decisions tu be made.

Ressarch applications of pS2 analysis are now emerging in the literature. This protein has been used as a marker of estrogenic activity for phytoestrogens and xenoestrogens, compounds with estrogen-like structures found in plant foods and in the environment (96,245).

In conclusion, we have described a simple, quantitative assay for pS2 protein and have applied it to study the mode of regulation of the pS2 gene by steroid hormones in the breast carcinoma cell line BT-474. Previously, we have shown that in breast cancer cell lines, the PSA gene is upregulated by androgens and progestins

(257). The combined use of pS2 and PSA as markers of hormonal activity in BT474 cells may altow us to assess the biological activity of natural and xenobiotic cornpounds which are currently considered as putative endocrine disruptors.

2.3 Is ICI 182,780 an Anti-Progestin in Addition to being an AntC Estrogen?

2.3.1 Introduction

ICI 182,780 (Faslodex) is a pure antiestrogen, used for the treatment of advanced breast cancer after failure of long-term adjuvant tamoxifen therapy (258,259).

Like other antiestrogens, ICI 182,780 may eventually find applications in othei aspects of breast cancer (e.g. prevention), as well as in other gynecological and nonrnalignant conditions. This compound is a derivative of estradiol, and as such, has high affinity for estrogen receptors (ER). However, because of its long side chain at the 7a position

(Figure 2.5), ICI 182,780 stearically hinders receptor dimerization. In the absence of dimerization, binding of the ER to estrogen response elernents (EREs) may be abolished or attenuated (260). ICI 182,780 has also been shown to cause the destruction of ER (261,262). It has therefore been demonstrated that ICI 182,780, in vitro mediates virtually no transcription of ER (263). ICI 182,780 Mifepristone (RU 486)

Figure 2.5, Structures of ICt 182,780 (faslodex) and RU 486 (mifepristone).

Other compounds that interact with or affect ER function are also under investigation or are already used in hast cancer therapy. These include the non- steroidal antiestrogens (NSAE), of which tamoxifen is the classic example. Second generations NSAE, which have greater antagonistic and lesser agonistic activity than tamoxifen (264-269), include raloxifene and drotoxifene. Progestins, such as FI4020 and antiprogestins, such as RU-486 (mifepristone) are also being examined.

Progestins are currently the most commonly used second-line endocrine therapy for advanced breast cancer (263). Antiprogestins have been in clinical trial for a few years

(263). However, the question remains as to their precise mechanism of inhibiting receptor-positive turnours.

Studies, using cells transfected with ER and PR reporter genes, have indicated one mechanism of action of antiprogestins. They dernostrate extensive inhibitory cross- reactivity between ER and PR. Both progestins (R5020) and antiprogestins (RU-486) have been shown to act as potent ligand-dependent repressors of ER activity when bound either isoform of PR (270). Moreover, RU-486 has been shown to stimulate growth of MCF-7 cells at 1O* M, which can be blocked by either 40H-tamoxifen and ICI

164,384, suggesting weak estrogenic activity (271). A recent study (272) has pointed to

"reversen cross-reactivity between these receptors, i.e. blocking of progestin-induced transcription by the antiestrogen ICI 182,780. In this study, cell lines devoid of endogenous hormone receptors were transfected with PR expression plasmids, and a luciferase reporter gene driven by progesterone response elements (PREs).

Reconstituted hormone action in cells devoid of endogenous receptors has advantages and disadvantages. Among the advantages is the simplicity of the system and the avoidance of "cross-talk" between various types of receptors. The incorporation of reporter genes with minimal promoter elements and hormone response elements

(HREs) further facilitates quantification of semi-quantitative detection of the response.

However, such systems may provide misleading or physiological non-relevant results, especially when multiple HREs are incorporated into the promoters of the reporter gene which then become 900-sensitiven reporters (273). Furtherrnore, physiologically relevant ratios of various hormone receptors, CO-activators,and CO-repressors and transcription factors are not present in cell lines naturally devoid of hormone receptors.

It would be important to verify newly identified actions of hormonal or antihormonal agents in artificial systems by using cells containing natural receptors and endogenous genes which are regulated by süch receptors. In this study we examine the recentIy identified antiprogestin action of ICI 182,780 by using the hormone receptor-positive breast carcinoma cell line BT-474 and the endogenously reguiated genes pS2 60

(estrogen-regulated), prostate specific antigen (PSA) (androgen-progestin-regulated) and human glandular kallikrein (hK2) (androgen-progestin-regulated).

2.3.2 Materials and Methods

2.3.2.1 Materials

The BT-474 human breast carcinoma ce !II line was obtained from the American

Type Culture Collection (ATCC), Rockville, MD. The levels of ER and PR are 29 and

389 fmol/mg, respectiveiy. Although at present AR cannot be quantified, we know from

Northern blot studies that this cell tine contains AR (274). ICI 182,780 was purchased from Tocris Cookson, Inc., Ballwin, MO, and RU-486 (mifepristone) was a gift from

Roussell UCLAF, Paris, France. All steroids used were obtained from Sigma Chemical

Co., St. Louis, MO, and stock solution were prepared at 109 M in anhydrous alcohol.

2.3.2.2 Methods

BT-474 cells were grown to confluency in phenol-free RPMl media (Gibco BRL,

Gaithersburg, MD) supplemented with 10% fetal calf serum, 10 rng/mL insulin and 200 mM L-glutamine at 37OC, 5% CO2. Once confluent, they were subcultured in 24-well microtiter plates using the same media, but with substitution of charcoal-stripped fetal calf senim for the regular fetal calf serum. The cells were then stimulated with either a steroid (progesterone, norgestrel or estradiol) alone at a concentration of 10" M, blocker (ICI 182,780 or RU-486) at 1og, IO-?,1 O" or IO-= Mlalone, or both blocker and steroid together. in wells with both blocker and steroid, blocker was added first, then incubated for 1 hour prior to addition of steroid at the conditions specified above. The plates were then incubated for 7 days, at which time the supernatants were harvested.

They were then analyzed quantitatively for pS2, prostate-specific antigen (PSA) and human glandular kallikrein (hW) as exemplified below.

Assays

pS2 Assay. We have used an ELISA-type competitive immunoassay for pS2 which was developed in-house. The details of this assay will be described elsewhere.

In short, we coat microtiter wells with a monoclonal anti-pS2 antibody and perfon a competition between a biotinylated 28-mer peptide derived from the N-terminal sequence of pS2 and endogenous pS2. After washing unreacted moieties, we detect

bound biotinylated peptide with a streptavidin-alkaline phosphatase conjugate and tirne-

resolved fluorometry, essentially as described elsewhere (248). The detection timit of

this assay is -20 ngimL.

PSA Assay. Prostate specific antigen was quantified with an ELISA-type

irnmunofluorometric procedure essentially as described elsewhere (275). The detection

Iimit of this assay is -1 ng/L.

hK2 Assay. An ELISA-type immunofluorometric assay was also used. The

details of this assay have been described elsewhere (276). The detection lirnit of this

assay is -6 ng/L.

All rneasurements were performed in duplicate and al1 experiments described

were perforrned at least twice. 2.3.3 Results

pS2 is an estrogen-regulated gene and the concentration of pS2 in the tissue culture supernatants reflects estrogenic activity. Estradiol stimulates pS2 production in a dose-dependent manner at concentrations from 10" M down to IO-" M. Other steroids do not induce any pS2 upregulation (data not shown). Similarly, the concentration of PSA and hK2 in the tissue culture supernatants reflect

progestationaVandrogenic activity. Both progesterone and norgestrel (and in addition testosterone and dihydrotestosterone, data not shown) stimulate PSA and hK2

production in a dose-dependent manner, from lu5M down to IO-" M. Estrogens

upregulate these two genes, but only at concentrations higher than 10" M (data not

shown). All three endogenous reporter genes used (pS2, PSA, hK2) are secreted

proteins and we found that 1 week post-single stimulation provide the best quantitative

and reproducible data,

ICI 182,780 and mifepristone were tested at concentrations from 10" to 1 M

for estrogenic (pS2) and proyestational/androgenic activity (PSA and hK2). The data

are shown in Table 2.1. ICI 182,780 did not show any agonistic activity at these

concentrations for either receptor. This was as expected for a pure antiestrogen. RU-

486 showed no estrogenic activity, but some progestationaVandrogenic activity was

found (Table 2.1). This is in line with other reports of agonistic activity of this

antiprogestin (277). Our controls worked as expected, i.e. induction of pS2 by estradiol

and of PSA and hK2 by progesterone and about 100-fold more potently by the synthetic

progestin, norgestrel (Table 2.1 ). Table 2.1. Production of pS2, PSA and hK2 by Blockers and Steroids

Cornpourid Concentration pS2 ng/mL PSA ng/L hK2 nqiL [Cl 182,780 IO-' M c20 15 c6 ICI182,780 IO-~M c20 2 1 6 -ICI182,780 10-'M <20 27 8 ICI 182,780 1u8 M <20 23 11 RU 486 1o'~ M c20 617 1054 RU 486 lod M c20 1050 4800 RU 486 10" M <20 656 2428 RU 486 IO* M e20 434 600 Est radio1 10" M 202 49 8 Progesterone 1O* M <20 799 1943 Norgestrel 1O-' M <20 66253 162822

Blocking experiments demonstrated that ICI 182,780, at concentrations of 10.' M

to 10'' M, showed 97-100% blocking of estradiol action. At 10.' M, this blocking was

minimal (only 16%). Interestingly, RU-486 showed 74-82% blocking of ER at al1

concentrations used (Figure 2.6). There was no dose-response for this effect,

suggesting that the phenomenon does not operate directly through the estrogen

receptor but by an alternative pathway, likely involving a cross-talk with the

progesterone receptor.

RU-486 showed complete blocking of PR, measured through both PSA and hK2,

when either progesterone or norgestrel was used as the progestin. For hk2, however,

mifepristone decreased blocking ability at IO*? M and beelw (Figures 2.7 and 2.8). This

was not seen for PSA, where blocking was 100% throughout (Figures 2.9 and 2.10). ICI 182,780 100 ,

O 1O* 1O-' 1o6 1O" Concentration of Blocker (M)

Figure 2.6. Percent Blocking of pS2 Production, by the Anti+strogen ICI 182,780 and Anti-progestin RU 486. 8T-474 cells were stimulated by 10" M estradiol. I- I-

Stimulant

ICI 182,780

Concentration of Blocker (M)

Figure 2.7. Percent Blocking of Human Glandular Kallikrein 2 (hK2) Production by ICI 182,780 and RU 486. BT-474 cells were stimulated by 10-~M progesterone. Stimulant 90 - Norgestrel10~M 80 70 t

60 -

50

40 30 - /

Concentration of Blocker (M)

Figure 2.8. Percent Blocking of hK2 Production by ICI 182,780 and RU 486. BT- 474 cells were stimulated by 1o4 M norgestrel. 100 . -I I - RU 486 90 -.

60

Concentration of Bkcker (M)

Figure 2.9. Percent Blocking of Prostate-Specific Antigen (PSA) Production by ICI 182,780 and RU 486. BT-474 cells were stimulated by 10'' M progesterone. RU 486 90 Stimulant 1 Norgestrel 10" M

1O" 1O" Concentration of Blocker (M)

Figure 2.1 0. Percent Blocking of PSA Production by ICI 182,780 and RU 486. BT- 474 cells were stimulated by 109 M norgestrel. 2.3.4 Discussion

The mechanism of gene regulation by steroid hormones is quite complex and involves many molecules including the steroid hormone receptors, heat shock proteins, transcriptional factors, CO-activators,CO-repressors, protein kinases, etc. (226,278,279).

The final result, regarding specificity and potency of response, will depend on the availability and concentration ratios of al1 these factors and may be heavily dependent on tissue type or, in in-vitro experiments, on type of cet1 iine used. Reconstituted steroid hormone action in cells that are devoid of hormone receptors is possible by exogenously transfecting plasmids containing the coding sequences of the receptors and plasmids containing minimal promoters with hormone response elements in front of indicator genes like CAT or luciferase. These simple systems have enjoyed widespread use because of their simplicity, ease of use and avoidance of complicating cross-talks between various receptor pathways. On the other hand, these systems should be approached with caution since they rnay produce results that have no relevance to the physiological situation. For example, a plasmid containing three or four EREs is progressively much more sensitive to estrogen stimulation than a plasmid containing only two EREs (273). Moreover, Weaver et al have reported, for example, that the antiestrogen ICI 164,384 had disparate actions on expression of the endogenous gene pS2 and on transfected reporters containing the pS2 estrogen response element (280).

Thus, caution should be exercised in interpreting data of reconstituted hormone action experiments until these are verified in cells which possess the steroid hormone receptor system and utilize endogenous genes as reporter systems. The study of Nawaz et al (272) is important since they report for the first time progestin-induced inhibition of transcription of the pure antiestrogen ICI 182,780. Thus, they conclude that ICI 182,780 acts as an antiprogestin with potency presurnably similar to that of the classical antiprogestin RU-486 (rnifepristone). They found no intrinsic progestin activity of 182,780 and no antiandrogen or antiglucocorticoid activity. The lCso of ICI 182,780 was around 2 x 1O-' M.

We set out to examine these data in a completely different experimental system.

Our cell line (BT-474 breast carcinoma cell line) had endogenous hormone receptors.

We also used endogenously regulated genes, i.e. pS2 (estrogen-regulated) and PSA and hK2 (androgen-progestin regulated). Quantitative analysis of the three secreted proteins in the tissue culture supernatants assures quantitative comparison of data.

The possible antipmgestational activity of ICI 182,780 and its comparison to that of RU-486 is important for three reasons. (a) This antiprogestational, in addition to its antiestrogenic activity of ICI 182,780 may be important in relation to its anti-cancer activity in-vivo; (b) This effect may allow us to better understand the clinical pharmacology of the drug and its possible therapeutic mechanisms, so that other compounds with similar activity are developed; (c) ICI 182,780 is used in in-vitro studies with cultured cells at concentrations around 10" M and the effects obsewed are attributed to its antiestrogenic activity. A possible antiprogestational activity of the drug at these levels will complicate the interpretation of data.

We have first verified that ICI 182,780 has no detectable estrogenic, androgenic or progestin activity even at concentrations as high as 10'' M (Table 2.1). Fumer, we have shown that RU486 has significant progestin agonist activity, as reported previously (248) and that norgestrel, a synthetic progestin, is about 100-fold more potent than progesterone, also in accordance with previous data regarding affinities of norgestrel and progesterone for the PR (248). In addition, we verified the specific upregulation of the pS2 gene by estradiol (Table 2.1).

The potent antiestrogenic activity of ICI 182,780 is shown in Figure 2.6. At 21 O-'

M, ICI 182,780 inhibited 197% of estradiol's action, as expected (Figure 2.6). We have further observed a strong antiestrogenic activity of the antiprogestin RU-486

(approximately 70-80% inhibition of estradiol's activity) which was not dose-responsive

(Figure 2.6). This activity does not seem to be directly related to the ability of RU-486 to block the estrogen receptor, but rather, to be an indirect phenornenon associated with progesterone receptors liganded by RU-486. Such an eifect has previously been published in the literature with progestins or antiprogestins demonstrating either inhibitory or stimulating estrogenic effects (270,271).

In Figure 2.7 we demonstrate, in agreement with the data of Nawaz et al, weak antiprogestational activity of ICI 182,780. The lCsoof ICI 182,780 for progesterone is between lu6- lo5 MI also in agreement with the previous report. However, the inhibition of the synthetic progestin norgestrel was only between 40-70% at an ICI

182,780 concentration of IO-' M and not detectable at lower concentrations.

Cornparison of the inhibiting (antiprogestational) activity of ICI 182,780 to RU486 clearly indicates that the latter is at least a 1000-fold more potent antiprogestin (Figures

2.7,2.8, 2.9 and 2.10).

ln conclusion, we have verified with a tissue culture system that contains endogenous receptors and with utilization of endogenous genes that indeed, ICI 182,780 has weak antiprogestational activity. However, this activity is far less than the activity of the synthetic antiprogestin RU-486. It seems unlikely that ICI 182,780 in-vivo, and at the concentrations used to treat breast cancer, will have any clinically significant antiprogestational activity in addition to its potent antiestrogenic activity. The weak antiprogestational activity of ICI 182,780 may complicate interpretations of tissue culture experiments in which the compound is used at concentrations ?log M and is considered a pure antiestrogen. CHAPTER 3 STEROID HORMONE ACTlVlTY OF FLAVONOIDS AND RELATED COMPOUNDS CHAPTER 3: STEROID HORMONE ACTlVlfY OF FLAVONOIDS AND RELATED COMPOUNDS

3.1 Rationale

After developing a tissue culture bioassay system to determine steroid hormone activities, Our next objective was to determine the agonist and antagonist activities of pure isoflavones, other flavonoids, and structurally-related compounds. Our secondary objective was to relate activity of these compounds to structural components, including importance of the flavonoid diaryl nucleus, position of the B ring, and hydroxyl side groups. Dr. Vigi Bemada of lndofine Chemical Co., Sommerville, NJ, a Company that specializes in natural compounds, kindly provided us with 60 flavonoids and related compounds, including the soy isoflavones biochanin A, genistein and daidzein. Another

15 compounds were purchased from Sigma Co., St. Louis, MO.

Because of the relatively large number of compounds, we first screened them at concentrations of 10" M and 1 O*' M for estrogenic and androgenidprogestational activity. The compounds that were found to be positive for activity were then tested in a dose-response manner al concentration of 10" to 10~M. The strongest estrogenic compounds were the soy isoflavones, genistein and biochanin A. The majority of the compounds that had activity were flavonoids, and had hydroxyl groups at positions 7, 6 or 4'. A few flavonoids also had progestational activity, with apigenin having the strongest. All of these had hydroxyl groups at postions 7 and 4'. None of the soy isoflavones possessed progestational activity, demonstrating that position of the B ring on carbon 2 was necessary.

Antagonist experiments were conducted sirnilariy. The compounds were first screened at a concentration of IO* M. Antagonist activity was defined as the flavonoid inhibiting production of pS2 or PSA by greater than 50% when stimulated by a steroid

(1O-' M estradiol or DHT, or 1O-'' M norgestrel). Compounds that demonstrated at least

50% blocking were then tested for dose-response activity at 1O-= to 10*~M. ICI 182,780. rnifepristone and nilutamide (antiandrogen) were tested similady as controls. The most significant result of this study is that several of the cornpounds tested have antiandrogen activity. The initial observation was with the soy isoflavone genistein, which, until the scientific correspondence was submitted, was thought only as a potent phytoestrogen. Since then, at least one other study has confirmed this result (108).

We have demonstrated that several flavonoids were able ta inhibit PSA production in the BT-474 breast cancer and in PC-3(ARI2 prostate cancer cell lines.

As potential natural antiandrogens, genistein and other flavonoids may play important roles in prostate cancer prevention and treatment. Research in this field has only started being pursued, and many more in vitro and in vivo studies must be conducted, for clinical relevance to be determined.

3.2 Agonist Activity of Flavonoids and Related Compounds on Steroid Hormone Receptors

3.2.1 Introduction

Many studies have focused on the soy isoflavones, particularly daidzein and genistein, and their possibie anticarcinogenic properties. These compounds, have been demonstrated in-vitro and in-vivo to have estrogenic as well as antiestrogenic activity

(83,971. Moreover, these compounds, have been shown in-vivo to lengthen the follicular phase of the menstrual cycle (191), reduce un'nary excretion of 17fbestradiol, and favor 2-hydroxyestrone formation (281), al1 of which are associated with reduced risk for breast cancer. More recently, there is increased interest on selective estrogen receptor modulators (SERMs) which hold promise of being used to prevent osteoporosis in post-menopausal women, and at the same time reduce the risk of developing breast cancer and atherosclerosis (282-285). Raloxifene is one SERM that is now in clinical trials (286). Others have put fonrvard the idea that phytoestrogens may represent natural SERMs (287).

lsoflavones are members of a larger family of compounds, known as the flavonoids. This family also includes flavones, flavanones, and flavans, which, Iike isoflavones, contain a phenyl side-chain, with a variable number of hydroxyl or other groups (Figure 1.1). These compounds have some structural similarities to the natural estrogen estradiol, as well as other steroid hormones and steroid hormone antagonists

(Figure 1.2).

The study of flavonoids and their possible role in preventing chronic diseases including heart disease, and breast and prostate cancers, has increased dramatically in the last decade. These compounds exist in relatively large amounts in Our food supply, especially in fruits and vegetables, tea, red wine, and legumes. Intakes of these compounds are now estirnated to be up to 45 mglday (288), and a database of foods with known flavonoid content has been created (67).

In-vitro, flavonoids have been shown to act as antioxidants (289), arrest the cell cycle and inhibit topoisornerase Il (290,291), and exert antiproliferative activities (292-

295). Many of the obsewed effects are thought to play a role in preventing breast andior prostate cancers. With the exception of soy isoflavones, few studies have focused on the estrogenicity of the flavonoids per se (102,103,296), and to date, to our knowledge, only one study has examined the possible androgenic and progestational activities of flavonoids (297). Furthermore, examination of flavonoid structure and correlation to biological activity (e.g. estrogenicity) has been limited (101,298). Whether similar structure/function relationships exist for estrogen, androgen and progesterone receptor binding of these phytochemicals is unknown.

Therefore, we assessed the estrogenic and androgenic/progestational activities of 72 flavonoids and related compounds using a human breast cancer cell line system, utilizing two steroid hormone-regulated endogenous proteins. pS2 is an estrogen- regulated protein, and prostate-specific antigen (PSA) is regulated by androgens and progestins. We have developed immunoassays to measure these secreted proteins in tissue culture supernatants. The data allowed us to identify active cornpounds and compare activity to structure. These data may be useful for comparing relative potencies of each of the phytochemicals regarding their ability to bind and activate steroid hormone receptors.

3.2.2 Materlals and Methods

3.2.2.1 Materials

The BT-474 human breast cancer cell line was purchased from the American

Type Culture Collection, Rockville, MD. The levels of estrogen receptor (ER) and progesterone receptor (PR), as quantified by commercial ELISA assays (Abbott

Diagnostics, Abbot Park, Chicago, IL) were 29 and 389 fmoümg, respectively. Although the androgen receptor (AR) content was not quantified, Northern blot studies indicated that this ceIl line contains AR (299). Al1 steroids used were from Sigma Chernicai Co., St. Louis, MO, USA. Stock, lo9 M solutions of steroids were prepared in absolute ethanol. Flavonoids and related compounds were obtained from lndofine Chemical Co.,

Inc., Summerville, NJ, and Sigma. We prepared lu2M stock solutions in absolute ethanol. Mifepristone (RU 486) and nilutamide (RU 56187) were gifts from Roussel-

UCUF, Romainville, France.

3.2.2.2 Methods

BT-474 cells were grown to confluency in phenol-free RPMl media (Gibco BRL,

Gaithersburg, MD) supplemented with 10% fetal calf serum, 10 mg/mL insulin and 200 mM L-glutamine at 37OC, 5% COn. Once confluent, they were subcultured in 24-well microtiter plates using the same media, but with substitution of charcoal-stripped fetaf calf serum for the regular fetal calf serum.

The celfs were then stimulated with a flavonoid or related compound at 10' M and IO-^ M (final concentrations) and incubated for 7 days at the same conditions as above. Estradiol, norgestrel and dihydrotestosterone (DHT) at 10" M were used as positive controls and ethanol (solvent) as a negative control. After 7 days, the supernatants were harvested and analyzed for pS2 and PSA. Compounds found to stimulate production of pS2 or PSA were then tested for dose-response, in the range from 1u5 to 1 M. Assavs

pS2 Assay. pS2 protein was analyzed using an ELISA-type competitive fluorometric immunoassay. The details of this assay are described elsewhere (300).

The detection limit is -20 ng/mL.

PSA Assay. Prostate specific antigen was quantified with an ELISA-type immunofluorometric procedure essentialiy as described elsewhere (250). The detection limit of this assay is -1 ng/L.

3.2.3 Results

The breast carcinoma cell line ET-474 is steroid hormone receptor-positive. Hall et al have demonstrated presence of estrogen (ER), androgen (AR) and progestercne

(PR) receptor mRNAs in BT-474 cells (299). We have further confirmed presence of

ER and PR by ELISA assays and of AR by immunohistochemistry (data not shown).

Our tissue culture system is based on the principle that the selected endogenous genes pS2 and PSA are directly up-regulated by steroid hormones. The pS2 gene contains estrogen response elements in its proximal promoter and its expression is increased after estrogen induction (251), but not after androgen or progestin induction

(300). Similady, the PSA gene contains multiple androgen response elements in its promoterlenhancer region (301) and is up-regulated by androgens and progestins but not estrogens (257). Thus, this tissue culture system is a sensitive indicator of hormone receptor activation and induction of transcription. The principles of transcriptional activation by steroid hormones havé been described by Beato et a1 (84). Of the 72 flavonoids and related compounds tested (Table 3.1, Appendix), 18 were found to have estrogenic activity. Six compounds of this subset were also found to have androgenic andior progestational activity. Of these, one compound had strong activity (apigenin), two had significantly lower activity (fisetin, naringenin) and three had very weak activity (Table 3.1). The soy isoflavones, biochanin A, genistein and daidzein, demonstrated strong estrogenic activity; the same was true for luteolin and naringenin, two citrus flavonoids. Resveratrol, a trihydroxystilbene found in red wine, 6- bromo-2-naphthol, several hydroxyflavanones and two hydroxyflavones had moderate estrogenic activity (Table 3.1). A dose-response relationship for the three soy isoflavones is shown in Figure 3.1. Detectable estrogenic activity was seen at isoflavone concentrations around IO-' -lu8 M. The estrogenic activity of the cornpounds of Table 3.1 below naringenin was detectable only at a concentration of 1a"

M.

The relatively large number of compounds tested allowed us to correlate the structure of these compounds with the potency of their activity. Below, we sumrnarize out general observations. Table 3.1. Flavonoids and Related Comgounds With and Without Steroid Hormone Agonist Activity at 10 M

Compound with Estrogenic Activity Relative Acîivity (%) Genistein 100 Biochanin A 95 Luteolin 58 Oaidzein 55 Naringenin 40 7-Hydroxyflavone 25 6-Hydroxyflavanone 23 - Resveratrol 22 6-Bromo-2-naphthol 20 Chrysin 18 Apigenin 16 6-Hydroxyf lavone 15

- . Quercetin 1O 7,8-Dihydroxyflavone 8 4'-Hydroxyflavanone 8 7-Hydroxyflavanone 8 Compounds with Progestational Activity Relative Activity (%) Apigenin 1O0 Fiset in 1 0.8 - - Naringenin 0.7 Ch rysin 0.2 Luteolin 0.2 Morin 0.1 Compounds without Agonisî Activity Abrine 3,4-Dimethoxycinnarnic acid Mangostine Andrographolide 7,8-Oimethoxyflavone 5-Methoxyflavanone Ascorbic acid Ellagic acid 6-Methoxyflavanone 5,6-Benzoflavone Embelin 7-Methoxyflavanone 7,8-Benzoflavone Flavanone 2'-Methoxyflavone Bixin Flavone 4'-Met hoxyflavone

4'-Bromoacetophenone . Folic acid 5-Methoxyflavone Caffeic acid Gallic acid 6-Methoxyflavone Caff eine Gardenin 7-Methoxyflavone Chlorogenic acid Harmaline 6-Methylflavone Conessine Harmalol L-Mimosine Curcumine Hamine Naringin 2,4'-Dihydroxyawtophenone Hannol Picrotin 2,S-Dihydroxyacetophenone 7-Hydr oxyfiavan Piperine 2.6'-Dihydroxyacetophenone 2'-Hydroxyflavanone Ponqarnd 2,s-Dihydroxy-1,4-benzoquinone 3-Hydroxyflavone Rutin 2',6'-Dimethoxyacetophenone 5Hydroxyflavone Salicyfic acid 2,3-Dimethoxybentaldehyde Kataniin Theophyiline Figure 3.1. Estrogenic Activity of Three Isoflavones, Measured as ng of pS2 proteinlml in the Tissue Culture Supernatant. The activity was dose-dependent at the concentrations shown. 1. The flavonoid core structure (diaryi ring) is important for estrogenic and

progestational activity. From the 18 identified active compounds (Table

3.1), 16 were flavonoids. Of the 31 non-flavonoids tested, only 2 showed

weak estrogenic activity. These two compounds were a naphthol

derivative (6-bromo-2-naphthol), and a trihydroxystilbene (resveratrol). All

of the compounds demonstrating progestationaVandrogenic activity were

f Iavonoids.

2. Hydroxyi groups appear to be crucial for activity. If hydroxyl groups are

not present in the structure, no estrogenic or progestationaVandrogenic

activity is obsewed. For example, we observed no activity for 5,6-

benzoftavone, 7,8-benzoflavone, flavone and flavanone, while we

detected activity for 7-hydroxyflavone, &hydroxyflavone, 7,8-

dihydroxyflavone, 6-hydroxyflavanone, 4'-hydroxyflavanone, and 7-

hydroxyfkvanone.

3. When hydroxyl groups are methylated, activity is diminished. Examples

include 7,8-dimethoxyflavone, which has no estrogenic activity, versus

7,8-dihydroxyflavone (active); 7-methoxyflavone (inactive) versus 7-

hydroxyflavone (active), and 8methoxyflavone (inactive) versus 6-

hydroxyfiavone (active). However, this observation did not hold true for

biochanin A which is as potent as genistein, despite having a methoxy

group in position 3'. 4. Isoflavones, in general, were found to be more potent estrogens than

other flavonoids. The isoflavones Biochanin A, genistein, and daidzein

showed estrogenic activity in a dose-dependent manner, from IO-'M to

1o'~ M (Figure 3.1). The other flavonoids had no activity below 106 M.

Additionally, apigenin (a flavone) and genistein (an isoflavone) differ only

in the position of the benzyl ring (3 position versus 2 position). This

difference reduces apigenin's estrogenic activity by 84% in cornparison to

genistein.

5. Flavones and flavanones appear to have greater estrogenic potency than

flavans (e.g .7-hydroxyflavone > 7-hydroxyflavanone >> 7-hydroxyflavan).

6. The position of the hydroxyl groups appears to be important. Hydroxyl

groups at positions 6, 7 or 4' confer more potent estrogenic activity than

hydroxyls at positions 3, 5 or 2'. For example, 6-hydroxyflavone>3-

hydroxyflavone and 5-hydroxyflavone (inactive); 6-hydroxyflavanone>2'-

hydroxyflavanone (inactive); 7-hydroxyflavone>3-hydroxyflavone and 5-

hydroxyflavone (inactive); 7-hydroxyflavanone>2'hydroxyf lavano ne

(inactive) and 4'-hydroxyflavanone>2'-hydroxyflavanone (inactive).

Remarkably, the top 6 compounds with estrogenic activity (Table 3.1)

have a 7-position hydroxyl group and apigenin, the most potent

progestational agent also possesses a hydroxyl group at position 7.

Additionally, conjugation of the hydroxyl group at position 7 renders the conjugate inactive, e.g. naringenin (active) versus naringin (inactive). See

also point #3 above.

7. The most potent estrogenic compounds have between 2 and 4 hydroxyl

groups. At least one is localized in the 7 position of ring A, and another

one in the 4' position of ring B (e.g. 7,8-dihydroxyflavone is much less

potent than daidzein, in which the second hydroxyl group is in the 4'

position).

8. A hydroxyl or other group at position 3 or 8 interferes with agonist activity

on estrogen and progesterone receptors (e.g. luteolin> quercetin and rutin;

luteolin, apigenimfisetin; 7-hydroxyflavone>7,8-dihydroxyflavone}.

9. Specificity of flavonoids for estrogen or progesteronelandrogen receptors

is dependent on the position of the 6 ring (e.g. apigenin has

progestational activity versus genistein which is an estrogen). Frorn al1

compounds that have progestationaYandrogenic activity, none is an

isoflavone; al1 are either flavones or flavanones (Table 3.1). The

importance of the 4'-hydroxyl on progestational activity is further

demonstrated by comparing apigenin versus chrysin, apigenin versus

luteolin (which has an extra hydroxyl) and apigenin versus marin (has two

extra hydroxyls). 10. A double bond between carbons 2 and 3 is important for progestin activity

(0.g. apigenin versus naringenin). A single bond at this site reduces

activity by 99%.

3.2.4 Discussion

Flavonoids, as components of Our diets, have been demonstrated to have protective effects on heart disease (302,303), cancer (304), and other conditions and diseases (305-307). The cardioprotective eff ects of isoflavones are thought to be primarily due to their estrogen-like activity. Like estrogen replacement therapy, soy isoftavones have been demonstrated to increase arterial cornpliance (308).

Additionally, these compounds diminish the symptoms of menopause, including hot flashes (309). For these reasons, many women are reanalyzing the risks and benefits of hormone replacement therapy, and supplementing their diets with soy products instead. Commercial vendors have made available isoflavone tablets. Phytoestrogens are increasingly becoming a natural alternative to synthetic estrogens.

Other flavonoids (flavones, flavanones) have been examined mostly for their antioxidant capabilities. The Seven-Country Study was one of the first to link flavonoid consumption with decreased risk of heart disease (73). Phenolics found in red wine are often used to partially explain the "French Paradox" (31 0); antioxidants and flavonoids have been shown to reduce LDL oxidation (31 1). Moreover, their estrogenic capaciiy may be partially responsible for the increase in HDL, often observed in red wine drinkers (3t2). These two activities rnay work simuItaneously, providing the consumer with extra cardioprotective benefits. However, the issue of the "French Paradox" still remains controversial.

The role of flavonoids in cancer development, especially breast and prostate carcinomas has yet to be fully examined. Few epidemiological studies have been conducted (70). In-vitro studies show the antiproliferative, antioxidant and cytostatic effects flavonoids (289-293). Currently, herbal alternatives to dnigs, including PC-

SPES for prostate cancer management, are being sought (313,314). These products have been shown to be antiproliferative and antitumorigenic (315). However, the mechanism is not well understood, and controlled, prospective studies have not been conducted.

We have examined the estrogenic and progestationaVandrogenic activities of 72 flavonoids and related compounds in a human breast carcinoma cell line, by monitoring endogenously regulated genes (pS2 and PSA) in the presence of endogenous hormone receptors. Eighteen of the 72 flavonoids and related compounds showed estrogenic andlor progestational activity. The soy isoflavones demonstrated activity down to 10"-

10" M. However, most of the other flavonoids did not show activity beyond 10" M.

These concentrations are approximately 10-fold higher than those reported by other groups, including Miksicek (101) and Le Bail et al (102). Their studies used transfected estrogen-responsive reporter plasmid systems, measured through chioramphenicol acyl transferase or luciferase enzymes. Our system, using endogenous genes and receptors, may be closer to the in-vivo situation, in cornparison to data generated with transfected indicator genes and receptors. These synthetic systems are usually too sensitive and may give distorted results. This issue was stressed recently by Xu et al.

(315) for studies related to hormone receptor function.

We analyzed the structure of compounds with and without estrogenic and progestational activity and devised general guidelines, as has been done by Lien et al for phenolic antioxidants (316). Through the testing of a large number of compounds, we were able to establish the relative importance of flavonoid class (flavone, flavanone, isoflavone, flavan), the nümber and position of hydroxyl groups and double bonds on the biological activity of these cornpounds. It is clear that subtle changes in the structure of these compounds can bring about a large change in their biological activity and receptor specificity. Our results concur with those of Miksicek (101) and Le Bail et al (102) regarding the importance of the diaryl ring structure, as 16 of the 18 estrogenic compounds were flavonoids. Only resverattol (a trihydroxystilbene) and 6-bromo-2- naphthol exhibited weak estmgenic activity at a concentration of 1 M. Similarly, we agree with the importance of the 7 and 4' hydroxyl positions. However, we found the 6 hydroxyl position to confer estrogenic activity as well (6-hydroxyflavone, 6- hydroxyflavanone). Similar to other studies, we found that a hydroxyl group at position

3 or 8, or a total of pater than 4 hydroxyl groups reduces the estrogenic activity of the flavonoid. Hydroxyl groups at positions 2' or 5 did not significantly affect estrogenic activity, but methylation at the 7 or 4' position (with the exception of biochanin A) decreased estrogenic activity.

We have also examined the structure/function relationship of flavonoids and related compounds for progestationaVandrogenic activity. All of the compounds with such activity were Ravonoids, demonstrating that the diaryl ring is crticial. Similarly, they al1 had hydroxyl groups at positions 7 and 4'. Finally, the presence of a double bond belween carbons 2 and 3 appears to be highly important. Elimination of mis bond

(apigenin + naringenin) decreases this activity by 99%.

One of our significant observations is the specificity of the position of the 0 ring in relation to the flavonoid core; whether it is bound to position 2 or 3. Two compounds that have the same empirical formula and molecular weight, namely genistein and apigenin, display the strongest biological activity, one being estrogenic (genistein) and one progestational (apigenin). The only difference between these two compounds is the position of the 8 ring, which qualifies the cornpound as being either an isoflavone

(genistein) or a ftavone (apigenin). This structural difference may be expioited to develop compounds that target one receptor versus the other.

This study has demonstrated estrogenic and progestationaVandrogenic activity of several flavonoids and other compounds by using a screening systern based on a human breast carcinoma cell Iine. These activities should be taken into consideration to explain as to how these compounds, and the natural products in which they are found, rnay play a role in the prevention of breast andor prostate cancers. The identified stnicture/function relationships may aid in the development of drugs or derivatized natural products with greater activity and specificity. 3.3 Genlstein: A Potent Natural Anti-androgen

Flavonoids and other polyphenolic natural compounds have attracted much attention as candidate cancer preventive (317-31 9) and anticarcinogenic agents

(126,320,321), as well as antiatherogenic (73,303,308,322) and antioxidant compounds

(155,158,323). Although the public now consumes these compounds in significant amounts from dietary sources, food supplements, and, more recently, as "nutraceutical" tablets (308), their actual mode of action is not fully defined. The most compelling hypotheses correlate the biological action of flavonoids to their ability to mimic natural estrogens (83,99,324), like estradiol, or to act as antioxidants (158,323). Indeed, genistein, a natural soy isoflavone, is among the most potent known phytoestrogens.

The ability of flavonoids to act as androgen mimics or anti-androgens, has attracted much less attention. Recently, we have shown that apigenin, a natural flavone found in chamomile, olive leaves, and other plant sources, has potent progestational activity

(100,297).

We have examined the possible anti-androgenic activity of genistein using the steroid hormone receptor-positive breast cancer cell line BT-474. This cell line, when stimulated by dihydrotestosterone (DHT), produces prostate specific antigen (PSA) which is then secreted into the tissue culture supernatant and can be measured quantitatively by immunoassay. Details of this system are given elsewhere (250,297).

To study anti-androgenic activity, the cells are first exposed to the putative anti- androgen (l0-~-10~M) for 1 hour and then stimulated with DHT (1p M). Controls with only anti-androgen or only DHT were included in al1 experiments. Nilutamide was used as a control anti-androgen. Our data (Figure 3.2) clearly demonstrate potent anti- I 10 - I C 1 - Quercetin O' lob 1O-' 1O* 1 Concentration of Blocker (M)

Figure 3.2. Percent Blocking of DHT, as Measured by PSA Production, by Genistein, Quercetin and Nilutamide. BT-474 human breast cancer cells were grown to confluency in phenol-free RPMl media (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum, 10 mg/mL insulin and 200 mM L-glutamine at 37"C, 5% C02. Onc9 confluent, they were subcultured in 24-well microtiter plates using the same media, but with substitution of charcoal-stripped fetal calf serum for the regular fetal calf serum. The cells were stimulated with either candidate blocker (genistein, quercetin or nilutamide), blocker and steroid (DHT) or steroid alone. Blockers were tested at concentrations of 1U5 to 10" Ml and DHT was used at 10" M. For the cells stimulated with blocker and steroid, the blocker was added first, and incubated for one hour, then the cells were stimulated with steroid. Alcohol was used as a negative control. The plates were then incubated for 7 days, at which time the tissue supernatants were harvested (297). The supernatants were then analyzed quantitatively for PSA (250). Blocking activity was assessed by dividing the amount of PSA produced by the candidate blocker + steroid and by steroid alone and multiplying by 100. None of the candidate blockers induced any PSA production when added alone. androgenic activity of genistein, which is dose-dependent and is detectable down to lW7

M. Quercetin and a number of other flavonoids tested are devoid of such activity (data not shown).

These data clearly demonstrate for the first time that genistein exhibits potent anti-androgenic activity, in addition to its well-established estrogenic activity. Indeed, the therapeutic potential of this compound in prostate cancer patients may be related to its combined estrogenic and anti-androgenic properties. It will be interesting to examine large numbers of natural compounds for anti-androgenic activity, which may qualify them as candidate therapeutic and preventive agents for prostate, breast and possibly other hormonally-dependent cancers.

3.4 Flavonoids Can Block PSA Production by Breast and Prostate Cancer Cell Lines

3.4.1 Introduction

Prostatic carcinoma is the most commonly diagnosed cancer in North American men, and the second leading cause of cancer death in this population (1). The incidence of prostate cancer has risen sharply over the last decade (1,325-327). Much of this increase can be attributed to better screening techniques, including serum prostate-specific antigen (PSA) analysis (326,328). Other potential contributing factors include the aging population, increasing rates of obesity, high consumption of meat and fat, combined with reduced physical activity (329-332).

Since most prostate tumors are hormone-dependent, combined androgen blockade (CAB) is a common strategy for primary or secondary therapy (333-335). Agents used for treatment include diethylstilbestrol (an estrogen agonist) (336-3381, luteinizing hormone-releasing hormone (LHRH) agonists (339-341), and anti- androgens, including flutarnide, nilutamide and cyproterone acetate (342-345). Many of these drugs have side effects including gynecomastia, impotence and hepatic toxicity

(346-352). For these reasons, many patients are searching for "natural" alternatives or complernents to traditional drugs (313,353-355).

Over the past few years, the beneficial effects of soy and soy isoflavones in the prevention of breast and prostate cancers, as well as of heart disease have been investigated (96,108,289,319,356-358). A body of epidemiological evidence associates consumption of soy, and vegetables in general, with lower prostate cancer risk in Asian populations versus Western countries (319,356,358). In vitro studies have demonstrated the partial estrogen/anti-estrogen activities of soy isoflavones and other flavonoids (38,39), as well as their antioxidant effects (96,102). Cytostasis and induction of apoptosis are two mechanisms by which the monoterpene, perillyl alcohol

(found in citrus fruit), and the synthetic flavone flavopiridol may be effective against prostate and other cancer cells (360-364). These compounds are currently in phase II clinical trials (365,366).

To date, few studies have investigated the ability of plant-derived compounds to block androgenic activities, such as expression of androgen-dependent genes. The soy isoflavone, biochanin A, was demonstrated to decrease testosterone-induced production of PSA in the LNCaP prostate cancer cell line, through upregulation of a catabolic enzyme (367). Resveratrol, the phytoalexin found in red wine, has been shown to have estrogenic and anti-estrogenic activity (1 04,368) and to downregulate transcription of both androgen receptor (AR) and PSA genes in this cell line (109). We therefore underîook this study to determine whether various flavonoids and related compounds could inhibit PSA production, a protein regulated by androgens, through the

AR.

3.4.2 Materials and Methods

3.4.2.1 Materials

The BT-474 human breast cancer cet1 line was purchased from the American

Type Culture Collection (ATCC), Rockville, MD. The levels of ER and PR, as quantified

by commercial ELISA assays (Abbott Diagnostics, Abbott Park, Chicago, IL) were 29

and 389 fmoVmg protein, respectively. Although the AR content was not quantified,

Northern blot studies indicated that this cell line contains AR (299). The PC-3 cell line,

PCS(AR)2, transfected with the human full-length AR cDNA, was described elsewhere

(369). AR concentration of this cell line is 491 fmoümg protein. The parental line has

been described as having both glucocorîicoid and estrogen receptors, although the

latter is controversial (370-372). Al1 steroids used were from Sigma Chemical Co., St.

Louis, MO. Stock, 104 M solutions of steroids were prepared in absolute ethanol.

Flavonoids and structurally-related compounds were obtained from lndofine Chemical

Co. Inc., Summerville, NJ and Sigma. We prepared lu2 M stock solutions of these

compounds in absolute ethanol. Nilutamide (RU 56187) was a gift from Rousset-

UCLAF Romainville, France. 3.4.2.2 Methods

BT-474 cells were grown to c~nfluencyin phenol-free RPMl media (Gibm BRL,

Gaithersburg, MD) supplernented with 10% fetal calf serum, 10 mg/mL insulin and 200 mM L-glutamine at 37"C, 5% COn. Once confluent, they were subcultured in 24-well microtiter plates using the same media, but with substitution of charcoal-stripped fetal calf serum for the regular fetal calf serurn. PCS(AR)2 cells were grown under similar conditions, using supplernentation of media with 5% fetal calf serum and 100 pg/mL hygromycin-B (Calbiochem-Novabiochem Corp., La Jolla, CA).

BT-474 cells were incubated with a flavonoid or related compound at 1U5 M and

IO-' M (final concentrations) for t hour, after which tirne the cells were stimulated with dihydmtestosterone (DHT) at 10" M. The cells were then incubated for 7 days at the same conditions as above. DHT was also tested alone (no blacker added) to determine

maximum production of PSA, and candidate blackers were tested without steroid (DHT), to determine theC androgen agonist activity. The blocking activity of nilutamide (an established anti-androgen) used at IO*? M, served as a positive control for anti- androgen activity, and ethanol (solvent) was a negative control. After 7 days, the tissue culture supernatants were harvested and analyzed for PSA. Compounds found to have greater than 50% blocking activity of DHT-induced PSA production were then tested for dose-response activity, in Me range fmm 1 to 1O-' M in bath BT-474 and PC-3(AR)i cell lines. Estradid was also tested, at concentrations of ICI-' to lo4 Ml to detemine whether this steroid had blocking activity. Each experiment was repeated at Ieast twice, and cells were assayed using trypan bIue to rnonitor cell viability. Assavs

PSA Assay. PSA was quantified using an ELISA-type immunofluorometric procedure described elsewhere (250). The detection limit of this assay is -1 ng1L. Total protein for al1 supematants did not differ significantly (data not shown).

3.4.3 Results

Our tissue culture testing system is based on the principle that the selected endogenous gene, PSA, is up-regulated by steroid hormones, in this case, DHT. The

PSA gene contains multiple androgen response elements (ARES) in its promoterlenhancer region (301). After binding of the AR to its respective ligand, a conformational change occurs, and the ligand-receptor complex subsequently binds to these ARES of the PSA gene promoter. If other transcription factors are also available, the gene is transcribed to mRNA, and then translated to PSA protein, which is secreted.

If any of these steps is inhibited, PSA protein production and secretion decrease, and this will be reflected by the PSA concentration in the culture medium. Thus, this tissue culture system is a sensitive indicator of hormone receptor activation and induction of transcription. This system has been used successfully in gene regulation studies

(84,257,373) and in identifying biological activity of candidate synthetic progestins (374).

The principles of transcriptional activation by steroid hormones have been reviewed by

Beato et al (û4).

Inhibition of PSA prodution was defined as greater than 50% blocking of DHT- induced PSA production. Twenty-two of the 72 tested flavonoids and structurally- related compounds were found to significantly inhibit PSA production in the BT-474 breast cancer cell line (Table 3.2, Figure 3.3). These include the soy isoflavones, biochanin A and genistein, the red wine phytoalexin resveratrol, and naringenin, a flavanone found in citrus fruits. Eight of these compounds showed dose-response

blocking of PSA production from lu5down to 10" M (Figure 3.4). Eleven of the 22 compounds were previously demonstrated to have estrogen activity, while the other 11

were found to be non-estrogenic (100). Estradiol also blocked PSA production from 10' 'down to 1 M in the BT-474 cell line. In the PC-3(AR)2 clonal Iine, several of these compounds showed no significant

blocking of PSA production. These included genistein, biochanin A, chrysin, naringenin

and 6-hydroxyflavone. Similarly, estradio1 did not inhibit PSA production in this cell line

(Table 3.2). The compounds listed in Table 3.3 did not alter PSA production in any of

the two cell line systems.

3.4.4 Discussion

The purpose of this study was to evaluate inhibition of androgen-regulated gene

expression by flavonoids and structurally-related compounds, measured as inhibition of

DHT-induced PSA production in two ceIl line-based systems. Several studies have

investigated estrogen activity of similar compounds and natural products containing

these polyphenols (100,l 03,146). However, very few studies have examined

androgenic or antiandrogenic activities (104,109,110,367,368).

Our study has demonstrated that several flavonoids can inhibit PSA production,

including isoflavones (genistein, biochanin A), flavones (luteolin, chrysin), and

flavanones (naringenin), which were hydroxylated (5-hydroxyflavone, 2'- Table 3.2. Aavonoids and Related Compounds that Signiflcantly Inhibited PSA Production in the BT-474 and PC4(AR)2 Cell Lines

Compound' % Blocking of PSA % BlocWng of PSA Production in BT- Production in PC- 4742 3(AR)2 6-Bromo-2-naphthol* 1O0 1O0 2,s-Dimethoxyacetophenone* 100 99-1 00 Flavone 1O0 80-85 5-Hydroxyflavone 1O0 100 2'-Methoxyflavone 1O0 60-70 5-Methoxyflavone 1O0 1O0 Harmol 99-1 O0 80-85 4'-Methoxyflavone 99-1 00 100 2'-Hydroxyf lavanone 98-1 O0 90 Biochanin A* 98-95 c50 Mangostine 81 -1 00 1O0 7-Hydroxyf lavanone* 81-87 95 Curcumine 80-97 99- t 00 Genistein* 76-87 <50 4'-Hydroxyflavanone* 74 98-100 Resveratrol* 72-99 70-75 Harmalol 67-91 ~50 Naringenin* 65-67 40 6-Hydroxyflavone* 64-92 c50 Luteolin* 62-8 1 80-85 Chrysin* 57-91 55-60 6-Methoxyflavanone 55-89 90-95 Estradio13 80-90 c50

' Compounds tested at 1 M final concentration. Compounds tested at least twice. Blocking expressed as range, where applicable. Estfadi01 was tested at 10.' M. 'Compounds found to be estrogenic in a previous study (100). Table 3.3. Flavonoids and Related Compounds That Did Not lnhibit PSA Production

Abrine 7,8-Dihydroxyflavone 7-Hydroxyflavone

Andrographolide-. 2',6'- Karajin Dimethoxyacetophenone Apigenin 2,3- 5-Methoxyflavanone Dimethoxybenzaldehyde Ascorbic acid 3,4-Dimethoxycinnamic acid 7-Methoxyflavanone 5,6-Benzoflavone 7,8-Dimethoxyflavone 7-Methoxyflavone 7.8-Benzoflavone Ellaaic acid 6-Methvlflavone - Bixin EmbeÏin L-Mimosine 4'-Bromoacetophenone Fisetin Morin Caffeic acid Flavanone Naringin Caffeine Folic acid Picrotin Chlorogenic acid Gallic acid Piperine Conessine Gardenin Pongamol Daidzein Hanaline Quercetin 2',4'-Dihydroxyacetophenone Harmine Rutin 2' ,5'-Dihydroxyacetophenone 7-Hydroxyflavan Salicylic acid 2',6'-Dihydroxyacetophenone 8Hydroxyflavanone Theophylline 2,5-Dihydroxy-1,4- 3-Hydroxyflavone benzoquinone Figure 3.3. Dose-Response Inhibition of PSA Production of Flavonoids and Related Compounds in the BT-474 Human Breast Cancer Cell Line. Significant inhibition is shown as percent blocking of DHT-induced PSA production. Compounds were tested at lo9 - la5 M, estradio1 was tested at 10'' - IO-' M, and DHT was used at 10" M. 2',5'-DMAP = 2',5'-dimethoxyacetophenone. hydroxyflavanone), methylated (6-methoxyflavanone, 5'-methoxyflavone) or neither

(flavone). Other compounds that showed significant PSA-blocking include nitrogen- containing (6-bromo-2-naphthol harmalol, harmol), and carboxyl-containing (curcumine,

2'5'-dimethoxyacetophenone) phenols. Several of these compounds were shown to have estrogenic activity in a previous study (100).

The ability of flavonoids and other polyphenols in regulating androgen-regulated proteins is not well studied. Three recent papers have al1 used the human prostate cancer cell line LNCaP, derived from the lymph node metastasis of a prostate cancer patient. The study by Mitchell et al (109) demonstrated dramatic decreases in androgen-induced PSA and human glandular kallikrein (hm) production in the presence of resveratrol (50-150 PM). Our data supports this finding, but we used much lower resveratrol concentrations (10 PM). Using a PSA-promoter fragment in front of a luciferase reporter gene, they showed that this phytoalexin abolished androgenic induction of the PSA promoter, resulting in significant decreases in transcription of both

AR and androgen-regulated genes.

Similar results were seen with green tea polyphenols (GTP) (1 10). 20-60 pglmL

GTP, incubated with LNCaP cells 1 hour prior to treatment with testosterone, was found to significantly reduce production of ornithine decarboxylase, another androgen- regulated protein. Moreover, Northern blot analysis revealed that this alrnost complete inhibition occurred at the transcriptional level. Moreover, in vivo, when GTP was added to drinking water, at 0.2% wlv, a 40% inhibition of omithine aecarboxylase activity was found in castrated and sham-operated rats, compared to controls. These two studies indicate that anti-androgen activity of these compounds must occu r through inhibition of transcription of AR ancilor androgen-controlled genes, blocking of the receptor, or a combination of these events.

A study conducted with flavonoids, also in LNCaP cells, found that biochanin A significantly induced activity of UDP-glucuronyl transferase, an enzyme responsible for metabolizing testosterone to inactive products. Through this mechanisrn, inhibition of

PSA production was also found (367). In this study a slight increase in binding sites of the AR, and AR protein was noted, therefore, inhibition of AR transcription was not a plausible mechznism for the action of these flavonoids.

In conclusion, we found that several flavonoids and related compounds can significantly inhibit PSA production, an androgen-regulated protein. These plant-derived compounds may thus play a role in the prevention andior management of prostate cancer. CHAPTER 4: STEROID HORMONE ACTlVlTY OF NATURAL PRODUCTS AND NUTRACEUTICALS CHAPTER 4: STEROID HORMONE ACTlVlTV OF NATURAL PRODUCTS AND NUTRACEUTCALS

4.1 Rationale

After determining the agonist and antagonist steroid hormone activities of pure compounds, the next objective was to evaluate such activities of natural products and nutraceuticals. These were al1 natural or processed products claimed to contain flavonoids and related compounds previously tested.

With the perception of a deteriorating health care system, coupled with

increasing access to medical information via the media and the internet, many patients

and healthy individuals are turning to natural products and over-the-counter

nutraceuticals to treat or prevent diseases including breast and prostate cancers, and

cardiovascular disease. Many claims have been made for these herbs, seeds, roots

and their processed products. These include hormonal and anticarcinogenic activities.

Soy extracts have gained much popularity as a source of natural estrogens and

progestins, for use in menopausal women to suppress symptoms including hot flushes,

and to prevent osteoporosis and/or heart disease. Other isoflavone preparations,

including red clover extract is also being used for these reasons. Saw palmetto has

been scientifically proven to aid in the treatment of urinary problems associated with

benign prostatic hypertrophy (BPH), and is now being clinically tested for use in prostate

cancer.

This study was undertaken to determine whethet sources of isoflavones, i.e. red

clover and soy extracts, demonstrate steroid hormone activity in the tissue culture

system similar to observations made with the pure isoflavones. Moreover, this study was to evaluate steroid hormone activity of other commonly used natural products and nutraceuticals that contain several of the flavonoids tested in the previous studies.

4.2 Effects of Natural Products and Nutraceuticals on Steroid Hormone- Regulated Gene Expression

4.2.1 Introduction

Recently, there is a trend for healthy individuals and cancer patients to use complementary ancilor alternative medicines (375). These are largely unproven therapies (356) which, at present, have no government regulations. Self-prescribed herbal and natural products represent one of the most popular alternative therapies used by the North American public (375,376). Moreover, the majority of households from the NHANES III study (377) admits taking herbal supplements, many without reporting such practices to physicians or other health care workers (376-378).

Healthy men and women, and prostate and breast cancer patients report several hormonally-related reasons for taking herbal rememdies. Saw palmetto (Serenoa serrulata), an extract from the saw palmetto berries, is commonly used for treating benign prostatic hypertrophy (BPH) and accompanying urinary tract problems (379-

381), and is now being extended, through self-medication, to prostate cancer management (356). Pumpkin extract is also being used for BPH (379). Dong quai

(Angelica sinesis) and Mexican wild yam root (Dioscorea villosa) are being used by menopausal and post-menopausal women as phytoestrogens andfor phytoprogestins

(382-384). lsoflavone preparations are being sold on the market (e.g. PromensiP by

Novogen, Estro-LogicTMby Quest) to reduce risk of cardiovascular disease (205,386- 388), osteoporosis (387,388) and to combat menopausal symptoms such as hot flushes

(389,390).

Other nutraceuticals being widely used by the public include echinacea

(Echinacea purpurea, Echinacea angustifolia) to boost immune function (391,392),

Saint. John's Wort (Hypericum perforatum) as an anti-depressant (393,394), cranberry extract for antibacterial and antioxidant activity (395,396), and grapeseed extract, for its antioxidant activity (397,398). Many of these natural products have been hypothesized to have anticarcinogenic activity (397-399), and are being used ad libitum by breast and prostate cancer patients (400,401).

Because many of these natural products presumably have hormonal activity, and

others are being used by individuals with, or at risk of developing hormone-dependent

cancers, we have undertaken this study to evaluate steroid hormone agonist and

antagonist activities of these preparations, using an in-vitro tissue culture system. The

BT-474 human breast cancer cell line, which is positive for estrogen (ER), androgen

(AR) and progesterone (PR) receptors, was used. For assessing biological activity, we

utilized secreted proteins, namely pS2, which is estrogen-regulated, and prostate

specific antigen (PSA), which is under the control of androgens and progestins.

4.2.2 Materials and Methods

4.2.2.1 Materials

The BT-474 human breast cancer cell Iine was purchased from the American

Type Culture Collection (Rockville, MD). The levels of ER and PR, as quantified by

commercial ELISA assays (Abbot Diagnostics, Abbot Park, Chicago, IL) were 29 and 389 fmol/mg protein, respectively. Although the AR content was not quantified,

Northern blot studies indicated that this cell line contains AR (299). All steroids used were from Sigma Chernical Co. (St. Louis, MO). Stock, IO'* M solutions of steroids were prepared in absolute ethanol. Natural products and nutraceuticals were purchased from pharmacies and health food stores (Table 4.1), with the exception of Estro-Logicm, which was a gift from Bruce Chapman, Boehringer lngelheim Self Medication. ICI

182,780 was purchased from Tocris Cookson, Inc., Ballwin, MO, and RU-486

(mifepristone), and Nilutamide (RU 56187) were gifts from Roussel-UCLAF

(Romainville, France). Stock, 10" M solutions of antagonists were prepared in absolute ethanol.

4.2.2.2 Methods

Preparations of Natural Products and Nutraceuticals

All liquid extracts were diluted 1:10 in anhydrous ethanol. Contents of capsules were mixed with 2 mL ethanol and incubated overnight. The liquid phase was then separated by centrifugation. Gelcaps were broken and the liquid contents were diluted with 1 mL ethanol. Tablets were crushed and incubated with 2 mL ethanol overnight. The liquid layer was then separated by centrifugation. More dilute solutions were further prepared in ethanol. Table 4.1. Natural Products and Nutraceuticals Tested

Name Active Form Source 1 ComponentIClaim Black Tea Theaflavins Tea Natural food store Canadian Ginseng Ginsenosides Capsule Natural food store Carob Syrup B vitamins Syrup Natural food store Chamomile Apigenin Gel-tab Jamieson Natural Sources Cran-Max Procyanidins Tablet Swiss Natural Sources Dong Quai Phyto-progestin Extract Swiss Natural Sources Echinacea Immune function, anti- Extract Swiss Natural Sources inflarnmatory Estro-Logic Soy isoflavones Capsule Quest Evening- Primrose y-linolenic acid Gel-tab Jamieson Natural Oil Sources Garlic Allium extract Gel-tab Swiss Natural Sources Grapeseed Procyanidins Capsule Swiss Natural Sources Green Tea Catechins Tea Natural food store Promensil Red clover isoflavones tablet Novogen Ltd. Prostate-Ease Saw palmette, flaxseed, Gel-tab Swiss Natural Sources pumpkin seed extracts Rosehips Vitarnin C Tablet Swiss Natural Sources Saw Palmetto Alleviate BPH symptoms Extract Herbs Etc. Siberian Ginseng Ginsenosides Capsule Natural food store St. John's Wort Anti-depressant Capsule Swiss Natural Sources Waterrnelon Juice Prostate health Juice Natural food store Wild Yam Rad Diosaenin Ext ract Gaia Herbs Inc. Definition of Bioloaical Activity

In this study, we have used the breast carcinoma cell line ET-474 as a biological testing system and the steroid hormone-regulated genes pS2 (estrogen-regulated) and

PSA (androgen and progestin-regulated) as indicators of hormone action. Natural products that stimulated pS2 production were defined as having estrogenic agonist activity while these which stimulated PSA production were defined as having progestationaVandrogenic agonist activity. Discrimination between progestational and androgenic activity were made by blocking experiments with either mifepristone (anti- progestin) or nilutamide (anti-androgen). Blocking (antagonist) activity of the natural products was defined as their ability to block production of pS2 (stimulated by estradioi) or PSA (stimulated by either dihydrotestosterone or norgestrel). These definitions are descriptive of the final effect but do not imply any mechanistic aspects of this up or down regulation of the indicator genes.

Cell Culture

BT-474 cells were grown to confluency in phenol-free RPMl media (Gibco ERL,

Gaithersburg, MD) supplemented with 10% fetal calf serum, 10 rng/rnL insulin and 200 mM L-glutamine at 37OC, 5% CO2. Once confluent, they were subcultured in 24-well microtiter plates using the sarne media, but with substitution of charcoal-stripped fetal calf serum for the regular fetai calf serum. Aaonist Activitv Studv

The cells were stimulated with natural product at full strength (stock solution), and 10, 100 and 1000-fold dilutions. Estradiol, norgestrel and dihydrotestosterone

(DHT) at 10' M were used as positive controls and ethanol (solvent) as a negative control. The cells were incubated with the stimulant for 7 days. The tissue culture supematants were then harvested and quantified for pS2 and PSA proteins.

Preparations that were found to stimulate PSA production were subsequently tested using mifepristone (RU-486, an anti-progestin) or nilutamide (an anti-androgen) to determine whether the effect was progestational or androgenic.

Antaaonist Activitv Study

The BT-474 cells were incubated with natural product at full strength, or at 10,

100 or 1000-fold dilutions for 1 hour, after which time the cells were stimulated with either estradiol, norgestrel or DHT at 104 M. The cells were then incubated for 7 days at the same conditions as above. Steroid was also tested alone (no candidate blocker added) to determine maximum production of pS2 and PSA. The blocking activity of faslodex (ICI 182,780, an anti-estrogen), mifepristone and nilutamide used at 10-7 Ml served as positive controls for antagonist activity, and ethanol (solvent) was a negative control. After 7 days, the tissue culture supernatants were harvested and analyzed for pS2 and PSA. Percentage blocking was calculated by quantifying the concentration of pS2 or PSA produced by natural product + steroid, divided by the concentration of pS2 or PSA produced by steroid alone, and multiplying by 100. Products were defined as having antagonist activity if their blocking activity was 250%. Assavs

pS2 Assay. We use an ELISA-type competitive immunoassay for pS2 which was developed in-house. The details of this assay are described elsewhere (300). The detection limit of this assay is -20 ng/mL.

PSA Assay. PSA is quantified using an ELISA-type immunofluorometric procedure described elsewhere (250). The detection limit of this assay is -1 ng/L.

These methods have been useful in several studies we have conducted, using pure flavonoids and antiestrogens (100,402).

4.2.3 Results

Of the twenty products tested (Table 4.11, four demonstrated significant estrogenic activity at the highest concentration. These included the red clover isoflavone preparation, PromensilTM,the soy isoflavone preparation Estro-LogicTM, grapeseed extract, and chamomile extract. Data with the firçt two preparations are presented in Figure 4.1. PromensilTMexhibited cytotoxic activity in the undiluted form, similar to that observed with genistein at high concentrations (100). At Iower concentrations, these was a dose-response relationship and the estrogenic agonist activity was detectable up to 1000-fold dilution (Figure 4.1). Estro-LogicTMdid not have any cytotoxic effect at the dilutions tested, and also demonstrated a dose-response relationship down to 100-fold dilution (Figure 4.1). Promensit's activity was about 7 times higher than Estro-Logic's at their most potent dilutions. This activity was equivalent to estradioi's estrogenic activity at IO-' M. Promensil Estro-Logic

Figure 4.1. Dose-Response Estrogen Activity of Promensilm and Estro-LogicTM. Dose-response activity was demonstrated for both isoflavone preparations; red clover isoflavones and soy isoflavones, respectively. The produtcts were tested undiluted, and at 10, 100 and 1000-fold dilutions in anhydrous ethanol. The maximal activity seen with PromensiP (10-fold dilution) was equivalent to the activity of 1o'~ M estradiol (data not s hown). Grapeseed and chamornile extracts exhibited both weak estrogenic and weak

progestational activity but only at the highest concentrations tested (-200 ngImL pS2

concentration in tissue culture supernatant). None of the natural products tested

exhibited androgenic activity (as assessed by PSA production by the cell line BT-474).

On the other hand, several products exhibited anti-estrogenic and anti-androgenic

activity at the highest concentration tested (Table 4.2). These included Prostate-Ease,

wild yam root and dong quai (anti-estrogens) and rosehips, dong quai and PromensilTM

(anti-androgens). Table 4.2 summarizes the activities of the products that have been

tested positive with our screening method.

Table 4.2. Biological Activity of Natural Products as ûetected by Our Tissue Culture Screening Procedure

Product Activity ~gonist' ~ntagonisp Estrogen Progestin Androgen Estrogen Androgen Progestin PromensiPM x3 X (63%) Estro-LogicTM X Grapeseed extract X X Chamornile extract X X Prostate-Ease X (77%) Wild yam root X (70%) Donq quai X (50%) X (71%) Rosehi~s X (75%)

1. For dose-response curves see Figure 4.1. 2. In brackets we present % blocking under the specified experimental conditions. 3. X represents specified activity. 4.2.4 Discussion

The use of alternative and complementary products is rapidly expanding

(356,375-378). As the North American population ages, hormonal events such as menopause and andropause are coming to the forefront of medical issues (403,404), and the incidences of steroid hom~one-dependentcancers are continuing to increase

(1). Many menopausal women are turning to natural alternatives such as soy isoflavones, wild yarn root, and dong quai. Similarly, many men, are looking towards complementary or alternative therapies to treat BPH, or prevent prostate cancer

(314,400,405). Saw palmetto and PC-SPES are among many appealing alternatives

(314,379,381,405).

PromensilTMcontains 40 mg isoflavones per tablet, primarily genistein, biochanin

A, daidzein and fornononetin (406),which is approximately equivalent to the isoflavone content of 2 cups of soy milk, Isoftavones have been found to be increase arterial cornpliance in postmenopausal women (3081,thus, presumably reducing heart disease risk. Recently, it has alsi, been clinically shown to maintain bone density (407). Our findings suggest a mechanism for these physiological effects. This isoflavone preparation showed significant estrogenic activity, equivalent tu 10' M estradiol. This product also demonstrated anti-androgen activity, similar to that demonstrated for the soy isoflavones genistein and biochanin A in previous studies (1O6,l O?')

Grapeseed extract, which contains procyanidins, compounds structurally similar to flavonoids, and chamornile, which contains apigenin, a flavone, showed both estrogenic and progestational activity at the highest concentration tested. Procyanidins present in polyphenok fractions of grapeseeds are strong antioxidants, and have been demonstrated to have antiproliferative and anticarcinogenic activities in cell culture and animal studies, respectively (408-410). Apigenin has been shown in our previous studies to have weak estrogenic, and relatively strong progestational activities

(100,297). This flavonoid is present in charnomile extract (41 l), and the activity of this natural product (Table 4.2) is likely due to apigenin.

Positive results were not found for al1 natural products tested. Dong quai, which has been made into tonics by Chinese women for centuries (383), and wild yarn root, both used as sources of phytoestrogens and phytoprogestins, were found not to have significant estrogenic or progestational activity, but rather to have weak anti-estrogenic andor anti-androgenic activities. A study conducted by Zava et al (412) looking at

Mexican wild yarn products containing diosgenin, found that this product did not bind to either ER or PR, and, moreover, this steroid precursor is not rnetaboiized to a

progestational form in the human body. Whether any true steroid hormone-related physiological benefit can be derived frorn either of these products has yet to be

ascertained. At present, the one double-blinded clinical study looking at the effects of

dong quai in postmenopausal women has found no benefit over placebo in alleviating

menopausal symptoms (413).

Saw palmetto and prostate-ease, both used for prostatic pathologies, including

benign prostatic hypertrophy and prostate cancer, did not show androgenic nor anti-

androgenic activity. Prostate-ease did show weak anti-estrogen activity. For saw

palmetto, these results suggest that its effects are likely not mediated by the steroid

hormone receptor system. One suggested mechanism is anti-inflammatory, through al-

adrenoreceptors (414). In conclusion, we have demonstrated, by using an in-vitro system, that several natural products and nutraceuticals exhibit weak steroid-hormone agonist and antagonist activity, while many others do not. These data may be useful to explain some of the in-vitro biological activities or the lack of biological activity of these preparations. Moreover, the eventual precise of definition of the biological activities of these compounds may help in the more rational use of these as natural therapeutive or chemopreventive agents. CHAPTER 5: ANALYSES OF IN VIVO EFFECTS OF FUNCTIONAL FOOOS CHAPTER 5: ANALYSES OF IN VIVO EFFECTS OF FUNCTIONAL FOOOS

5.1 Rationale

After determining the in vitro steroid hormone activities of soy isoflavones in purified form and as an extract, it was important to examine their in vivo activities as part of functional foods. Therefore, this study was undertaken to determine the overall steroidal potential of biological fluids of subjects on diets high in this functional food.

The following study used 24-hour urines of subjects on one specific soy study. Several other studies have also been conducted, evaluating steroid hormone potential of soy or high vegetable feeding, using sera and urine, respectively. The data from the high vegetable study ("Simian Study") has recently been published (see List of Publications

Arising from this Work). Data from the other soy feeding studies are currently being analyzed.

In this work, biological fluids were not hydrolyzed prior to using them in the tissue culture system. In vivo, only -5% of isoflavones are found in the aglycone fom in the bloodstream and urine, with the large majority conjugated to glucuronides or sulfates

(415). The unconjugated isoflavones have been thought to be the more active components. However, this has not ahivays been shown to be the case. Morito et al have demonstrated that genistin, the conjugated form of genistein binds more weakly to

ER receptors and induces transcription less than genistein. However genistin stimulates the growth of MCF-7 cells more strongly than genistein, which may be important for carcinogenicity (41 6).

Hydrolysis prior to administration of the biological fluids in this system would have allowed for determination of total isoflavone activity, Le. in the aglycone fom. This has been done by others for chemical determination of isoflavone content of legumes and urine samples (417). Since the initial observations indicated that aglycone isoflavones are more active than the conjugated forms, hydrolysis would result in a gross overestimation of the biological activity of the circulating isoflavones. Therefore, in the present work, the samples tested were not hydrolyzed, and the ratios of ag1ycone:conjugated isoflavones were similar to those that human tissues would be exposed to in vivo. We consider our results to reflect more closely what occurs physiologically.

5.2 Effect of Soy Protein Foods on Low-Density Lipoprotein Oxidation and Ex Vivo Sex Hormone Receptor Activity

5.2.1 Introduction

lt has been suggested that soy food consumption may be part of the reason for the low rates of cardiovascular disease and hormone-dependent cancer in the industrialized nations of the Orient (418). Soy isoflavones have been proposed as the components of soy most responsible for many of the suggested health benefits (418-

421). They have been associated with the cholesterol-lowering action of soy (387,421 -

423), they have antioxidant activity in vitro (77',323,324,388,389), and, as

phytoestrogens, they have been suggested to play a possible role in reducing the risk of

breast and prostate cancer by a variety of mechanisms (96,245,418-421,424426)-

On the other hand, there are also those who believe that soy consumption poses

potential risks to health (427-433) and that, via excessive phytoestrogens activity, soy

consumption may enhance breast tumor growth, cause abnormal sexual development

in infants and children, and have adverse effects on the central nervous system (427- 433). Although there have been many studies on soy, many of these issues have not been dealt with specifically. In the meantime, the New Zealand Ministry of Health has issued a warning in relation to thyroid function in infants on soy formulated milk substitutes (434). On the other hand, as of October 1999, the US Food and Drug

Administration (FDA) has allowed health claims to be made for soy in regards to its lowering of serum cholesterol, a risk factor for cardiovascular disease (435).

5.2.2 Subiects and Methods

5.2.2.1 Subiects

Thirty-one hyperlipidemic subjects (19 men and 12 postmenopausal women) completed ho1 -month metabolic diet periods separated by at least a 2-week washout period in a randomized crossover study. The study details and data relating to serum iipids, blood pressure, fecal short-chain fatty acids and fecal bile acids are reported elsewhere (436). The subjects' mean age was 56.5 -+- 9.0 years (range, 31 to 70),with a body mass index of 24.6 r 2.3 kg.m3 (range, 20.8 to 29.1). All subjects had elevated serum low-density lipoprotein (LDL) cholesterol (4.1 mmoVL) and a triglycerides level less than 4.0 mmoVL at recruitment. None had clinical or biochemical evidence of diabetes or liver or renal disease and none were using hypolipidemic agents, with the exception of one man on lovastatin 20 mgld throughout the study. One woman was on hormone replacement therapy, 2 women were taking levothyroxine, and 1 man and 1 woman were using vitamin E supplements (400 to 800 mgld). Dosage levels of al! medications and supplements were held constant for both study periods. Subjects were also instructed to maintain their habitua1 level of physical activity throughout. Wood sarnples were obtained after a 12- to 14-hour overnight fast prior to the study and at the end of weeks 2 and 4 of each metabolic phase. Serum was stored at -70°Cprior to analysis. Body weight was measured at the start and at weekly intervals on both metabolic phases. Twenty-four-hour urine collections were made on an outpatient basis at the end of each phase.

The study was approved by the Ethics Committee of the University of Toronto and St. Michael's Hospital. lnformed consent was obtained from al1 subjects and their primary-care physicians.

5.2.2.2 Methods -Diets The rnetabolic diets were designed in conformity with the National Cholesterol

Education Program step 2 dietary principles, but the dietary intake of cholesterol was further reduced. The macronutrient profile of the metabolic diets on each phase is presented in Table 5.1. The test diet provided 86 mg isoflavones per 2,000 kcal daily, while no isoflavones were detected in the control diet (Table 5.2).

The test and control metabolic diets followed a 7-day rotating menu plan. The control diet was a lacto-ovo vegetarian diet with milk products that were low in fat, consisting of skim milk, 1% dairy fat yogurt, skim-miik cheese, and low-fat cottage cheese. Eggbeaters (Lipton's, Toronto, Ontario, Canada) were used rather than whole eggs to further reduce the cholesterol intake. fable 5.1. Calcutated Macronutrient lntake (mean f SE) on the Test and Control Metabolic Diets (Ndl) Nutrtent Control Test 1 kcaVd 2,519 186 2,341 188 MJ/d 10.5 I0.4 9.8 + 0.4 Total protein

Vegetable protein 9/d 27 + 1 110 I4 % 4.3 I 0.1 18.8 I 0.1 Soy protein S/d OIO 33 14 % 0.0 IO.0 5.7 f: 0.1 Available carbohydrate S/d 343 I12 318 + 12 % 54.6 I0.3 54.3 + 0.3 Total dietary fiber s/d 36I1 62 + 3 ! gJ1,OOO k~al 14.3 4 0.2 26.3 + 0.2 soluble fiber 4/d 9IO 18+1 gi1,000 kcal 3.4 IO.0 7.6 + 0.1 Total fat 1 , dd 71 43 66 +2 % 25.5 k 0.2 25.5 + 0.2 SFA s/d 18+1 16It

% 8.7 + 0.1 9.6 + 0.1 Dietary cholesterol mdd 7643 77 +4 mgfi ,000kcal 30.2 I0.4 33.0 & 1.4 Alcohol 4/d OIO OIO % 0.1 k0.1 0.1 kO.1 Abbreviations: SFA, saturated fatty acids, MUFA, monounsaturated fatty acids, PUFA, polyunsaturated fatty acids.

Table 5.2. lsoflavone Content of Study Diets (mean of seven lday 2,000 kcal composites)

lsoflavone (mgld) Control Test Daidzin - 16.8 + 2.3 Genistin - 33.7 + 4.7 Glycitin - 3.7 2 0.8 Malonyl daidzin - 11.3 2 3.6 Malonyl genistin - 13.4 + 6.0 Malonyl giycitin - 0.6 2 0.6 Daidzein - 3.3 + 0.6 [ Genistein - 1.6 2 0.8 Glycetin - 1.5 I0.7 Total daily intake 0.0 86.0 k 17.1 ln the test diet, 93% of the animal protein was replaced with vegetable protein from soy, other legumes, and cereal foods provided as easy-to-prepare meals or frozen dishes, meat substitutes, and vegetarian "cold cuts". Soluble fiber was increased by the inclusion of oats, barley, and legume dishes at breakfast cereals, soups and main dishes. The fatty acid profile and dietary cholesterol intake on both diets were balanced by inclusion of butter and whoie eggs on the test (e.g. 1 egg per week on a 2,000-kcaVd diet). Test food items used in this study were al1 readily available and were obtained from either a supermarket roo Good To Be True; Loblaw Brands, Toronto, Ontario; and Yves Veggie Cuisine, Vancouver, British Columbia, Canada) or a heatth food store

(MGM Products, Cedar Lake, MI; and Fantastic Foods, Petaluma, CA).

We have reported the details of the dietary and analytical methodology previously (436). The diets were balanced for fatty acids and plant sterols, since these might alter serum lipids and antioxidant status. At each dinic visit, the dietitian assessed cornpliance using the menus. Subjects were asked to weigh al1 foods and to check them against the menu plan when eaten. Additional items were noted in a blank column opposite the prescribed diet. These data were used to calculate the dietary intake (Table 5.1). Body weight was measured at each clinic visit, and the results were used to adjust the total caloric intake. Complete diets were packed at a central location and delivered weekly by courier to each subject's home at a time convenient to them. Analyses

We assessed estrogenic and androgenic activity in an ex vivo human tissue culture system using immunoassays for pS2 and prostate-specific antigen (PSA) produced after stimulation with the test materials (300,437). BT-474 breast cancer cells were grown to confluence and then subcultured in 24-well microtiter plates. Once they were confluent in the wells, the cells were stimulated with urine diluted 1:1,000 or estradiol or dihydrotestosterone (DHT) at IO-' M, the latter two serving as standards.

Anhydrous ethanol was used as a negative control. The plates were incubated for 7 days, and estrogenic activity was measured in the supernatant as the pS2 concentration using an immunoradiometric assay (CIS Bio. Gif-sur-Yvette, Cedex, France). pS2 is an estrogen-regulated protein that is demonstrated in the literature to be a good marker for estrogenic activity (243). Androgenic activity was measured using an enzyme-Iinked immunosorbent assay for PSA, an androgen-regulated protein (257). The procedure for this assay has been described elsewhere (257,437). pS2 and PSA concentrations were converted to equivalents of estradiol and DHT, respectively, using pS2 and PSA values obtained in the same assay for the steroid standards. Final values were expressed in picomoles.

Dietary isoflavonoid levels were measured in freeze-dried 24-hour composites of the 7-day test and control diets by high-performance Iiquid chromatography (HPLC)

(417,438) using a 600E multisolvent delivery system with a photodiode array detector monitoring at 200 to 350 nm (Waters. Marlborough, MA) and a YMC-pack-ODS-AM

303 column (5 Fm, 250 mm x 4.6 mm ID; YMC. Wilmington, NC) equipped with an AM direct-connect Cl8 guard column. Appropriate isoflavone standards were analyzed (Table 5.2). Biochanin A was used as an internai standard with recovery values of 80 to

100%. Urinary isoflavone levels were measured by HPLC after acid hydrolysis (417).

Chromatographs were obtained from the 3-dimensional array using a photodiode array detector at 258 nrn to allow an assessrnent of the common regions of relatively high absorbante for daidrein, genistein and the added recovery standard, flavone.

Statistical Analvsis

The results are expressed as the rnean _+ SE. The weight change is expressed as kilograms per month. The percentage treatment differences between the endpoint values for both diets were calculateci for each subject, and the data were assessed by

Student's t test (2-tailed) for paired data. A 2-sample t test was used for cornparison of treatment effects between subgroups. The absoiute difference between treatments was assessed using the General Linear Model procedure and SAS software (PROC

GLMfSAS) (439) with the end-of-treatment value as the response variable and the following main effects: diet, sex, treatment order (sequence), diet x sex, sex x sequence, a random term representing the subject nested within the sex x sequence interaction, and the baseline value as a covariate where measured. Pearson correlation coefficients were used to assess the significance of linear associations (439). A subject group of 30 altowed detection of a 28% difference in urinary ex vivo estrogenic activity

(assuming a 52% SD of the effect; with a=0.05 and p=0.8) (439). 5.2.3 Results

Of 31 subjects, 16 received the test diet first. The diets were well accepted and compliance was good. On the test diet, subjects consumed 95% + 7% of the calories provided. The respective control figure was 96% I6%. The test diet provided 86 mg isoflavones per 2,000-kcal diet daily, with no isoflavones detectable in the control diet

(Table 5.2). There was a significant weight gain over the 1 month of the control diet (0.2

+ 0.1 kg, P=0.043) and a nonsignificant weight loss over the 1month of the test diet (0.1

+ 0.2 kg, P=0.484). The treatment difference approached significance (0.3 + 0.2 kg,

P=0.069).

5.2.3.1 Urinarv lsoflavone Excretion

The 24-hour urinary volume on the test diet (1.84 + 0.1 7 L) and control diet (2.01 + 0.14 L) was similar. On the test diet, 0.8 + 0.2 mg/d genistein and 3.0 + 0.6 mgid were excreted in the urine. On the control diet, no isoflavonoids were excreted in the

urine except for very low levels of daidzein in one woman (0.1 mgid) who had the

highest level on the test diet (5.3 mg/d).

5.2.3.2 Ex Vivo Sex Hormone Rece~torActivity

No significant difference was found between treatment in urinary sex hormone

receptor activity either before or after creatinine adjustment, although the mean values

on the test diet tended to be lower than the control diet (Figure 5.1). On the control diet,

urinary estrogen equivalents were higher for women than men after creatinine

correction (7.5 I2.0 versus 3.9 + 0.6, P= 0.045). This difference between the sexes Test Contml Test

Control Test

Figure 5.1. Creatinine-corrected Urinary Estmgen (A) and Androgen Equivalents (B) (pmoUmmol) in Men (1149) and Postmenopausal Women (1142) on Test and Control Diets. was no longer significant on the test, due to a reduction in estrogen levels in women

(Figure 5.1).

5.2.4 Discussion

Our data indicate that readily available soy foods providing modest levels of soy protein reduce the indices of oxidized LDL without increasing urinary estrogen activity.

This supports previous reports that soy isoflavone consumption may protect LDL cholesterol from copper-mediated oxidative damage in vitro (388,389). However, in previous studies, no direct measurements were reported on unmodified LDL (388,389).

This study therefore demonstrates that an additional cardioprotective effect of soy in reducing oxidized LDL cholesterol levels at a dose of soy protein that reduces serum cholesterol and would qualify for an FDA cardiovascular disease nsk reduction health claim. These effects were achieved with no increase in urinary estrogen activity. Our data therefore do not support prior concerns about the possible increased breast cancer risk in women or aitered sexuai development in children consuming soy at these levels

(427-430).

Oxidized LDL is more readily taken up by the marcrophages of the scavenger system in the arterial wall and may contribute to plaque formation (440,441). The consumption of antioxidant flavonoids in tea, fruit and vegetables, lycopene in tomato products and vitamin E as a supplement have al1 been associated with a reduced risk of coronary heart disease (442-445). Vitamin E and lycopene have powerful antioxidant properties, reducing LDL oxidation and oxidative damage to plasma proteins (442,443). LDL concentrations were reduced on the soy-containing diets (436). Both the amino acid composition of soy proteins and their isoflavone content have been suggested to be responsible for the cholesterol-lowering action of soy

(386,422,423,444,445), although not al1 studies have reported an isoflavone effect

(308,446). Soy contains many other potentially active components, including saponins, plant sterols, and polyunsaturated fatty acids, ail of which may contribute to cholesterol reduction. In the present study, the Iipids and sterols were separated from the soy protein isolate used in the majority of the soy products consumed. Soy fatty acids and sterols were therefore unlikely to play a part in the lipid changes (436).

It is well recognized that plant-derived sex hormone analogs may have important physiological effects (191,447). However, despite the high plasma levels which may be achieved, compared with endogenous hormones, isoflavones have relatively low potency. Of even greater importance, they may act both as potential agonists and antagonists (418). Evidence for their blocking action includes studies in which soy isoflavones fed to young women resulted in significant lengthening of the menstrual cycle (191). In Our study, the ratio of dietary isoflavones to protein of 2.6 mglg was associated with a relatively low urinary output of genistein and daidzein (448) and a tendency, on soy, for a reduction in ex vivo sex hormone activity in the urine. These data indicate that major hormonal changes may not be found at modest levels of soy protein intake that reduce the risk factors for cardiovascular disease (436). Therefore, in relation to breast cancer (230,429), the concems about unwanted increases in estrogenic activity at the moderate levels of soy intake appear unwarranted.

Conversely, the tendency for sex hormone activity to be depressed on soy diets is in line with current strategies to treat and possibly prevent hormone-dependent cancers with sex hormone-blocking agents (449,450).

We conclude that a moderate intake of soy foods reduces the concentration of oxidized LDL cholesterol, possibly due to the increased consumption of isoflavonoids associated with soy protein. These changes were achieved without a significant alteration in urinary ex vivo hormone activity, which has been a major concern for those who predict potentially harmful effects from soy food consumption. Currently, soy consumption is advocated based on its cholesterol-lowering ability alone (422).

However, in view of the apparent success of antioxidant agents in preventing experimental arteriosclerosis (451) and the effect of dietary antioxidants in reducing the risk of cardiovascular disease in cohort studies (442,4431, the antioxidant effect of soy may add to its potential value in coronary heart disease risk reduction with no apparent risk of adverse effects. CHAPTER 6: GENERAL DISCUSSION CHAPTER 6: GENERAL DISCUSSION

6.1 General Discussion

Several epiderniological studies have shown an inverse association between soy consurnption and breast and prostate cancer incidences (452-455). Because most are ecological studies, examining risk across countries and regions, mechanism cannot be elucidated from them. Therefore, several in vitro, animal, and a handful of human studies have been conducted to examine various componentç of soy, and mechanisms through which it rnay be chemopreventive (191,192,456-458).

Several researchers have put forth theories that not only rnay soy not be beneficial in prevention of cancers, but rnay actually increase risk. Warnings have been given to mothers intending to use soy-based formulas that such feeding rnay huit their babies several decades down the road (434). Others are concerned that soy consumption by men and women who are at high risk of prostate or breast cancer rnay further increase this risk (428,429). The main hypothesis behind this is that because the soy isoflavones genistein, daidtein and biochanin A are estrogenic, they rnay increase overall steroid hormonal activities in vivo, thus increasing cancer risk.

Therefore, the purpose of this project was to assess the steroid hormone potential of plant foods, with special reference to soy. This was done by examining soy isoflavones in three forms, pure compound, plant extract (nutraceutical), and whole

(functional) food, and determining how the in vitro activities rnay translate in vivo to overall steroid hormone potential.

Steroid hormone activity throughout this project was defined as the ability to stimulate or inhibit estrogen- or androgenlprogestin- regulated proteins, Le. pS2 andor PSA in the tissue culture system we developed. Although direct binding of ER, PR or

AR was not done, previous work conducted in our lab has shown that the proteins measured are not produced in cell lines (breast and prostate) that do not possess these

receptors (302). Moreover, measuring estrogen-regulated pS2 has been correlated with direct ER binding (96).

After developing a tissue culture system and an assay to measure the estrogen-

regulated protein pS2, soy isoflavones and other flavonoids were evaluated for steroid

hormone activities, including estrogenic, progestational and androgenic. Of al1 compounds tested, genistein and biochanin A demonstrated highest estrogenic

activities, which were dose-responsive down to 109 M and 10" M. respectively. Soy

isoflavones did not possess progestational or androgenic activities. However, they did

demonstrated significant anti-androgen activity (76% and 98%, for genistein and

biochanin A, respectively at 10" M), defined as blocking dihydrotestosterone (DHT)-

stimulated prostate specific antigen (PSA) production. As soy has been associated with

reduction of prostate cancer risk (455), these results showed one potential mechanism

of action.

Soy and red clover (another source of the "soy isoflavones') extracts Sere then

tested in this system, as were several other natural products and nutraceuticals

commonly used by the public for supposed steroid hormone potential. As with pure

isoflavones, the commercial soy and red clover extracts had high estrogenic activities.

The estrogenicity of the highest strength of red clover extract used was equivalent in the

pS2 assay to that of estradiol at loa M. The estrogenic activity of soy extract was

significantly lower than red clover. Theoretically this would be more beneficial, as reducing overall estrogenicity is the mechanism by which soy isoflavones are believed to be related to breast cancer prevention (191,192,457).

Soy extract also demonstrated 63% anti-androgen activity at the highest strength tested. This is similar to the result seen with pure genistein, and supports prostate cancer prevention data.

The final objective of this project was to determine how the in vitro data may translate in vivo to overall steroid hormone potential. By using biological fluids obtained from individuals in a soy feeding study in our tissue culture system, we found that short- term feeding did not increase agonist potentials. These results indicate that soy consurnption does not increase overall estrogenic or androgenic potential in humans.

Moreover, we found non-significant reductions in estrogenic and androgenic activities in women and men, respectively. Because the sarnple population was small (19 men and

12 women), the power was not high enough to reach statistical significance. However, these results may indicate that long-term feeding of this functional food may reduce risk factors for steroid-hormone dependent cancers, which corroborate with epidemiological studies (452-455), as well as several prospective trials looking at risk factors for breast cancer (191,192,457).

The observation that biochanin A had estrogenic activity similar to genistein in

Our system is not in agreement with results obtained by other groups, using direct binding and recombinant yeast assays (96,103,230). Furthermore, some of the structure-function relationships we determined do not coincide with those seen by others (103,230), such as the strong activity shown by biochanin A, which contains a methoxy group on C-4'. One possible expianation may be cellular metabolism. We did not measure flavonoid metabolism by the breast cancer cells used in this system, however, it is possible that conversion of some compounds to more active metabolites, e.g. biochanin A to genistein, could have taken place. This would also explain the highly estrogenic activities seen with the red clover and isoflavone preparations, which have been shown to need significant metabolism to become estrogenic (415).

We did not hydrolyze the biological fluids prior to using them in our.system.

Because 95% of soy isoflavones are conjugated in biological fluids (415), by not hydrolyzing the urines and sera, only -5% of isoflavones are free to act in our system.

However, our objective was to ascertain the biological implications of soy feeding in healthy subjects, Le. to examine the in vivo situation using an in vitro assay system.

Hence, hydrolyzing the samples to give the maximum arnount of free isoflavone was not the aim. Rather, our aim was to assess the steroid hormone activity of the soy isoflavones with a ratio of bound:free isoflavone similar to that seen in vivo. Therefore, the choice not to hydrolyze the biological fluids was one made early on, and the results must be interpreted in that context. Moreover, a recent study has shown that although the conjugated isoflavone genistin, binds more weakly to ER and induces transcription less than genistein, it stimulates growth of MCF-7 cells more than the aglycone isoflavone (416). More work is needed to determine the in vivo activities of conjugated isoflavones.

In conclusion, these results indicate that soy isoflavones and other flavonoids have steroid hormone activities. These activities may act in vivo to modulate potency of potentially carcinogenic endogenous hormones, thereby reducing overall steroid hormone potential. Further research is needed to determine the physiological importance of flavonoids in prevention andior management of hormone-dependent cancers, and, moreover how to make best use of natural products, nutraceuticals and functional foods in individuals at risk.

6.2 Summary

Through the studies described, the following objectives have been met:

1. Developrnent of several techniques and methods to evaluate steroid hormone

activities of pure compounds, products and metabolites wit hin biological fluids.

An ELISA for pS2 protein was established, and several cell lines were evaluated

for their usefulness in our tissue culture system. Antagonists used for treatment

of hormone-dependent cancers were determined to be good positive controls of

steroid hormone blocking in our system.

Several flavonoids were found to possess weak estrogenic and/progestational

activities in vitro. The soy isoflavones had only estrogenic activities. These

activities were structurally-related, and were dependent on presence of the diaryl

nucleus and position and number of hydroxyl groups. None of the compounds

tested had androgenic activities, but several had antiandrogenic effects, as

characterized by their abiIity to inhibit DHT-stimulated PSA production. No

structure-function relationship could be established for this activity, but the dose-

dependency of a few of the estrogenic flavonoids was similar to the antiandrogen

effects seen for estradiol in the BT-47'4 cell line. 3. Soy and red clover extracts, both sources of "soy isoflavones", and other natural

products and nutraceuticals were then tested for steroid hormone effects. Like

pure flavonoids, the isoflavone-containing products showed significant estrogenic

activities. The soy extract showed antiandrogenic activity similar to that seen

with genistein. This study showed that steroid hormone activities seen with pure

compounds are maintained in extracts of natural products.

4. Steroid hormone potential of the functional food, soy, was detemined in healthy

subjects. By using non-hydrolyzed biological fluids obtained from individuals in a

soy feeding study, it was found that short-term feeding did not significantly alter

steroid hormone activity, compared to controls. Non-significant reductions in

estrogenic and androgenic activities in women and men, respectively, may

indicate that long-terni feeding of this functional food may reduce risk factors for

steroid-hormone dependent cancers. This study demonstrated that in vitro

estrogenic activity did not translate into in vivo estrogenic activity, but instead,

soy may act physiologically in an antiestrogenic manner, to reduce overall

estrogenic potential.

6.3 Future Directions

Future directions in this field should include work into the use of natural products, and functional foods. Further investigation into the steroid hormone activity of natural products and nutraceuticals should be conducted. Because these products are relatively new, and are currently under no specific regulations, studies must be carried out to determine their activities in vitro, and in vivo. Aspects such as unwarranted effects and interactions should be investigated. Processed products should be tested for flavonoid content, with results available to the consumer. Moreover, best use of these products as alternative or complementary therapies in individuals at risk of steroid hormone-dependent cancers warrants further research.

The use of soy as a functional food, as well as other foods requires specific attention. This project has shown that soy does not increase overall steroid hormone potential in men and postrnenopausal women. However, more work rnust be done to further confirm this. Moreover, the use of soy in reducing estrogenic and androgenic activity in vivo is important in prevention of cancer. Human trials with endpoints including steroid hormone activities, serum PSA, prostatic intraepithelial neoplasia

(PIN), pS2, breast tissue proliferation, et cetera, should be conducted. Other foods including flaxseed, fruits and vegetables, and teas, which contain flavonoids and related compounds with steroid hormone activities should also be tested.

6.4 Conclusion

This project enabled for the examination one mechanism by which flavonoids, in general, and soy specifically, may act to prevent steroid hormone-dependent cancers.

Moreover, this study demonstrates that observations made in vitro cannot necessarily be translateci directly in vivo. Although soy isoflavones do have estrogenic activity, the physiological impact was not estrogenic, but in fact, showed an antiestrogenic tendency.

The antiandrogenic effects of these isoflavones, a surprising finding, did translate in vivo. How these results may be applied in the use of soy in functional foods andior nutraceuticals will be interesting to see.

In conclusion, this project has been challenging, interesting and exciting throughout. Many fruitful results have been produced, leading to further questions on the importance of flavonoids in prevention andlor management of hormone-dependent cancers, and the use of natural products, nutraceuticals and functional foods in individuals at risk. These data are a starting point for research on the use of soy as a functional food, and will hopefully lead towards understanding of how soy functions in humans, and how best to exploit these health benefits. CHAPTER 7: REFERENCES CHAPTER 7: REFERENCES

National Cancer lnstitute of Canada: Canadian Cancer Statistics 2000, Toronto, Canada 2000.

Hertog MG, Hollman PC, Katan MB, Kromhout D. lntake of potentially anticarcinogenic flavonoids and their determinants in adults in The Netherlands. Nutr Cancer 1993;20:21-9.

Hollman PC, Katan MB. Dietary flavonoids: intake, health effects and bioavailability. Food Chem Toxicol 1999;37:937-42.

Aldercreutz H. Lignans and phytoestrogens: possible preventative rote in cancer. In Proaress in Diet and Nutrition, P Rozen and C Horowitz (eds). Basel: Karger 1988:165-76.

Aldercreutz H. Western diet and Western disease: some hormonal and biochemical mechanisms and associations. Scan J Clin Lab lnvest 1990;50:3-23.

Messina MJ, Persky VI Setchell KD, Barnes S. Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr Cancer 1994;21:113-31.

Steinmetz KA, Potter JD. Food-group consumption and colon cancer in the Adelaide Case-Control Study, 1: vegetables and fruit. Int J Cancer t9W;S3:7ll-9.

Bjelke E. Case-control study in Nonvay. Scan J Gastrentol 1974;9:42-8.

Modan 0, Barell V, Lubin F, Modan Ml Greenberg RA, Graham S. Low-fiber intake as an etiologic factor in cancer of the colon. J Natl Cancer lnst 1975;55: 15-18.

Phillips RL. Role of life-style and dietary habits in risk of cancer among Seventh-Day Adventists. Cancer Res 1975;35:3Sl3-22.

Graham S, Dayal H, Swanson Ml Mittelman A, Wilkinson G. Diet in the epidemiology of cancer of the colon and rectum. J Natl Cancer lnst 1978;61:709-14.

Miller AB, Howe GR, Jain Ml Craib KJ, Harrison L. Food items and food groups are risk factors in a case-control study of diet and colo-rectal cancer. lnst J Cancer 1983;32:lSS-61. Tajima KI Tominaga S. Dietary habits and gastro-intestinal cancers: a comparative case-control study of stomach and large intestinal cancers in Nagoya Japan. Jpn J Cancer Res 1985;76:705-16.

Macquart-Moulin G, Riboli El Cornee J, Charnay B, Bethezene Pl Day N. Case-control study on colorectal cancer and diet in Marseilles. Int J Cancer 1986;38:183-91.

Kune S, Kune GA, Watson LF. Case-control study of dietary etiological factors: the Melbourne Colorectal Cancer Study. Nutr Cancer 1987;9:21-42.

Graham S, Marshall J, Haughey 6, Mittelman A, Swanson Ml Zielezny Ml Byers Tl Wilkinson G, West D. Dietary epidemiology of cancer of the colon in western New York. Am J Epidemiol 1988;128:490-503. ta Vecchia Cl Negri El Decarli A, DIAvanzo B, Gallotti L, Gentile A, Franceschi S. A case-control study of diet and colo-rectal cancer in northern Italy. Int J Cancer 1988;41:492-8.

Slattery ML, Sorenson AW, Mahoney AW, French TK, Kritchevsky Dl Street JC. Diet and colon cancer: assessment of risk by fiber type and food source. J Natf Cancer lnst 1988;80:1474-80.

Tuyns AJ, Kaaks R, Haelterman M. Colorectal cancer and the consumption of foods: a case-control study in Belgium. Nutr Cancer l988;ll:l89-2O4.

Lee HP, Gourley L, Duffy SW, Esteve JI Lee J, Day NE. Colorectal cancer and diet in an Asian population-case-control study among Singapore Chinese. Int J Cancer 1989;43:1007-16.

Bentilo El Obrador A, Stiggelbout A, Bosch FX, Mulet M, Munoz N, Kaldor J. A population-based case control study of colorectal cancer in Maiorca, 1: dietary factors. Int J Cancer 1990;45:69-76.

Hu J, Liu Y, Yu Y, Zhao T, Liu S, Wang Q. Diet and cancer of the colon and rectum: a case-control study in China. Int J Epidemiol 1991;20:362-7.

Bidoli El Franceschi S, Talamini R, Barra S, La Vecchia C. Food consumption and cancer of the colon and rectum in north-eastem Italy. Int J Cancer 1992;50:223-9.

Peters RK, Pike MC, Garabrant D, Mack TM. Diet and colon cancer in Los Angeles County, California. Cancer Causes Control 1992;3:457-74. lscovich JM, L'Abbe KA, Castelleto R, Calzona A, Bernello A, Chopita NA, Imehutzsky AC, Katdor J. Colon cancer in Argentia, 1: risk from intake of dietary items. Int J Cancer 1992;51:851-7.

Graham S, Marshall J, Mettlin C, Rzepka T, Nemoto T, Byers T. Diet in the epidemiology of breast cancer. Am J Epidemiol 1982;lI6:68-75.

Zemla B. The role of selected dietary elements in breast cancer risk among native and migrant populations in Poland. Nutr Cancer 1984;6:187-95.

Katsuoyanni K, Trichopoulos O, Boyle P, Xirouchaki E, Trichopoulou A, Lisseos B, Basilaros S, MacMahon B. Diet and breast cancer: a case-control study in Greece. Int J Cancer 1986;38:815-20.

La Vecchia C, Decarli A, Franceschi SIGentile A, Negri E, Parazzini F. Dietary factors and the risk of breast cancer. Nutr Cancer 1987;10:205-14.

Hislop TG, Kan L, Coldman AJ, Band PR, Brauer G. Influence of estrogen receptor status on dietary risk factors for breast cancer. Can Med Assoc J 1988; 138~424-30. lscovich JM, lscovich RB, Howe G, Shiboski S, Kaldor JM. A case-control study of diet and breast cancer in Argentina. Int J Cancer 1989;44:770-6.

Simard A, Vobecky J, Vobecky JS. Nutrition and life-style factors in fibrocystic disease and cancer of the breast. Cancer Detect Prev 1990;14:567-72.

Ewertz M, Gill C. Dietary factors and breast cancer risk in Denmark. Int J Cancer 1990;46:779-W.

Lee HP, Gourley L, Duffy SW, Esteve J, Lee 3, Day NE. Dietary effects on breast cancer risk in Singapore. Lancet 1991;337:1197-1200.

Richardson S, Gerber M, Cene S. The role of fat, animal protein and some vitamin consumption in breast cancer: a case-control study in southern France. Int J Cancer 1991;48:1-9.

Pawlega J. Breast cancer consumption and smoking, vodka drinking and dietary habits. Acta Oncol 1992;31:387-92.

Kato 1, Miura S, Kasumi F, Iwase T, Tashiro H, Fujita Y, Koyama H, Ikeda T, Fujiwara K, Saotome K, Asaishi K, Abe R, Nihel M, Ishida T, Yokoe Tl Yamamoto H, Murata M. A casecontrol study study of breast cancer among Japanese women: with special reference to family history and reproductive and dietary factors. Breast Cancer Res Treat 1992;24:51-4. Levi F, La Vecchia ClGulie Cl Negri E. Dietary factors and breast cancer risk in Vaud, Switzerland. Nutr Cancer l993;19:327-35.

Rohan TE. Dietary fiber, vitamins A, C and El and risk of breast cancer: a cohort study. Cancer Causes Control 1993;4:29-37.

Haenszel W, Berg JW, Segi Ml Kurihara M, Locke FB. Large-bowel cancer in Hawaiian Japanese. J Natl Cancer lnst 1973;51:1765-79.

Bjelke E. Case-control study in Minnesota. Scand J Gastroenterol 1974;9:49-53.

Martinez 1, Torres RI Frias Z, Colon JR, Fernandez N. Factors associated with adenocarcinomas of the large bowel in Puerto Rico. In Advances in Medical Oncoloay, Research and Education. Vol 3. Oxford, England: Pergamon Press 1979:45-52.

Manousos O, Day NE, Trichopoulos D, Gerovassilis F, Tzonou A, Polychronopoulou A. Diet and coIorectal cancer: a case-control study in Greece. Int J Cancer 1983;32:1-5.

Zaridze DlFilipchenko V, Kustov V, Serdyuk V, Duffy S. Diet and colorectal cancer: results of two case-control studies in Russia. Eur J Cancer 1993;29:112-5.

Phillips RL, Snowdon DA. Dietary relationships with fatal colorectal cancer among Seventh-Day Adventists. J Natl Cancer lnst 1985;74:307-17.

Thun MJ, Calle EE, Namboodiri MM, Flanders WD, Coates RJ, Byers Tl Boffette Pl Garfinkel L, Heath CW Jr. Risk factors for fatal colon cancer in a large prospective study. J Natl Cancer lnst 1992;84î:l49l-l5OO.

Steinmetz KA, Kushi LH, Bostick RM, Folsom AR, Potter JD. Vegetables, fruit and colon cancer in the Iowa Women's Health Study. Am J Epidemiol 1994;139:1-15.

La Vecchia Cl Decarli A, Fasoli MI Parrazini F, Franceschi S, Gentile A, Negri E. Dietary vitamin A and the risk of intraepithelial and invasive cenrical neoplasia. Gynecol Oncol 1988;30:187-95.

Marshall JR, Graham SI Byers Tl Swanson Ml Brasure J. Diet and smoking in the epidemiology of cancer of the cervix. J Natl Cancer lnst 1983;70:847- 51. La Vecchia ClFranceschi S, Decarli A, Gentile A, Fasoli MlPampallona S, Tognoni G. Dietary vitamin A and the ~skof invasive cervical cancer. Int J Cancer l984;34:319-22.

Verreault R, Chu JI Mandelson M, Shy K. A case-control study of diet and invasive cervical cancer. Int J Cancer 1989;43:1050-4.

Ziegler RG, Brinton LA, Reeves WC, Brenes MM, Tenorio F, de Britton RC, Gaitan E. A case-control study of nutrient status and invasive cervical cancer. Am J Epidemiol 1991;134: 1335-46.

Zemla B, Guminski LI Franek K, Koslosza 2, Banasik R. Etiological factors in invasive corpus uteri carcinoma. Neoplasma 1991;38:157-63.

La Vecchi ClDecarli A, Fasoli Ml Gentile A. Nutrition and diet in the etiology of endometrial cancer. Cancer 1986;57:1246-53.

Zemia BI Guminski S, Banasik R. Studies of risk factors in invasive cancer of the corpus uteri. Neoplasma 1986;33:621-9.

Barbone F, Austin Hl Partridge EE. Diet and endometrial cancer: a case- control study. Am J Epidemiol 1993;137:393-403.

Shu X-O, Zheng W, Potischman N, Brinton LA, Hatch MC, Gao TT, Faument JF Jr. A population-bassd case-control study of dietary factors and endometrial cancer in Shanghai, People's Republic of China. Am J Epidemiol 1993; 137:lSS-65.

Levi F, Franceschi S,Negri E, La Vecchia C. Dietary factors and the risk of endometriai cancer. Cancer 1993:71:3575-81.

Byers Tl Marshall J, Graham SIMettlin Cl Swanson M. A case-control study of dietary and non-dietary factors in ovarian cancer. J Nat1 Cancer lnst 1983;71:681-6.

La Vecchia Cl Decarli A, Negri El Parazzini F, Gentile A, Cecchetti G, Fasoli Ml Franceschi S. Dietary factors and the risk of epithelial ovarian cancer. J Natl Cancer lnst 1987;79:663-9.

Schuman LM, Mandel JS, Radke A, Seal U, Halberg F. Some selected features of the epiderniology of prostatic cancer: Minneapolis St. Paul, Minnesota case-control study, 1976-1979. In Trends in Cancer Incidence: Causes and Practical Information, Magnus K, ed. Washington, DC: Hemisphere Publishing Corp, 1982:345-54. Graham SI Haughey B, Marshall JI Priore R, Byers Tl Rzepka Tl Mettlin C, Pontes JE. Diet in the epidemiology of the prostate gland. J Nat1 Cancer lnst 1983;70:687-92.

Talamini R, La Vecchia Cl Decarli A, Negri El Franceschi S. Nutrition, social factors and prostatic cancer in a northern ltalian population. Br J Cancer 1986;53:817-21.

Le Marchand L, Hankin JH, Kolonel LN, Wilkens LR. Vegetable and fruit consumption in relation to prostate cancer risk in Hawaii: a reevaluation of the effect of dietary beta-carotene. Am J Epiderniol 1991;133:215-9.

Talamini RI Franceschi SI La Vecchia Cl Serraino Dl Barra SI Negri E. Diet and prostatic cancer. Nutr Cancer 1993;18:277-86.

Hsing AW, McLaughlin JK, Schuman LM, Bjelke El Gridley G, Wacholder S, Cochien HT, Blot WJ. Diet, tobacco use, and fatal prostate cancer: results from the Lutheran Brotherhood Cohort Study. Cancer Res 1990;50:6836-40.

Pillow PC, Duphome CM, Chang S, Contois JH, Strom SS et al. Developrnent of a database for assessing dietary phytoestrogen intake. Nutr Cancer l999;33:3-19.

Block G, Hartman A, Dresser Cl Carroll Ml Gannon J et al. A data-based approach to dietary questionnaire design and testing. Am J Epidemiol 1986; 124:453-69.

Block G, Coyle L, Hartman A, Scoppa S. Revision of dietary analysis software for the Health Habits and History Questionnaire. Am J Epidemiol 19W;l39:119O-6.

Strom SS, Yamamura Y, Duphorne CM, Spitz MR, Babaian RJ, Pillow PC, Hursting SD. Phytoestrogen intake and prostate cancer: a case-control study using a new database. Nutr Cancer 1999;33:20-5.

Hertog MG, Feskens El Hollman P, Katan MB, Krornhout D. Dietary flavonoids and cancer risk in the Zutphen Elderly Study. Nutr Cancer 1994;22:175-84.

Schurman A, Goldbohm A, Dorant El van der Brandt P. Vegetable and fruit consumption and prostate cancer nsk: a cohort study in The Netheriands. Cancer Epidemiol Biomarkers Prev 1998;7:673-80.

Hertog MG, Krornhout Dl Aravanis Cl Blackburn H, Buzina R et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the Seven Countries Study. Arch lntem Med 1W5;l 55:381-6. Knekt P, Jarvinen R, Seppanen R, Haliovaara Ml Teppo L et al. Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am J Epidemiol 1997; 146:223-30.

Shutt DA. The effect of plant oestrogens on animal reproduction. Endeavour 1976;35:110-3.

Verdeal K and Ryan DS. Naturally-occurring estrogens in plant foodstuffs: a review. J Food Protect 1979;42:577-83.

Adlercreutz CH, Goldin BR, Gorbach SL, Hockerstedt KA, Watanabe S, Hamalainen EK, Markkanen MH, Makela TH, Wahala KT, Adlercreutz T. Soybean phytoestrogen intake and cancer risk. J Nutr 1995; 125:757S-77OS.

Farnsworth NR, Bingel AS, Cordell GA, Crane FA, Fong HHS. Potential value of plants as sources of new antifertility agents. II. J Pharm Sci 1975;ô4:717-54.

Jordan VC, Mittal SI Gosden BI Kock RI Lieberman ME. Structure-activity relationships of estrogens. Environ Health Perspect 1985;61:97-llO.

Uehar M, Arai Y, Watanabe SI Adlercreutz H. Comparison of plasma and urinary phytoestrogens in Japanese and Finnish women by time-resolved fIuoroimmunoassay. Biofactors 2000;12:217-25.

Coward L, Kirk M, Albin N, Barnes S. Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry. Clin Chim Acta l996;247:121-42.

Watanabe S, Yamaguchi Ml Sobue T, Takahashi T, Miura T, Arai Y, Mazur W, Wahala KI Adlercreutz H. Pharrnacokinetics of soybean isoflavones in plasma, urine and feces of men after ingestion of 60 g baked soybean powder (kinako). J Nutr 1998 Oct;128:1710-5.

Wang TT, Sathyamoorthy N, Phang JM. Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis 1996;17:271-5.

Beato Ml Truss M, Chavez S. Control of transcription by steroid hormones. Ann N Y Acad Sci 1996;784:93-123.

Celio L, Bajetta El Toffolatti L, Catena L, Beretta E, Buttoni R. Ovarian ablation for premenopausal early-stage breast cancer: an update. Tumori 2OOO;86:1914. Walsh PC, Siiteri PK. Suppression of plasma androgens by spironolactone in castrated men with carcinoma of the prostate. J Urol 1975;114:254-6.

Bono AV, DiSilverio F, Robustelli della Cuna G, Benvenuti Cl Brausi M, Ferrari P, Gibba A, Galli L. Complete androgen' blockade versus chemical castration in advanced prostatic cancer: analysis of an ltalian multicentre study. ltalian Leuprorelin Group. Urol Int 1998;60:18-24.

Dalesio O. Complete androgen blockade in prostate cancer: an overview of randomized trials. Prostate Suppl 1992;4:llI-4.

Bono AV, Poui E, Robustelli della Cuna G, Preti P. Prostatic cancer - survey of hormonal treatment in Europe. J Int Med Res l990;18 Suppl 1:Il- 25.

Potischman N, Troisi R. In-utero and early life exposures in relation to risk of breast cancer. Cancer Causes Control 1999;10:561-73.

Sanderson M, Williams MA, Daling JR, Holt VL, Malone KE, Self SG, Moore DE. Maternal factors and breast cancer risk among young women. Paediatr Perinat Epidemiol 1998;12:397-407.

Herbst AL, Anderson D. Clear cell adenocarcinoma of the vagina and cervix secondary to intrauterine exposure to diethylstilbestrol. Semin Surg Oncol 1990;6:343-6.

Honnritz RI, Viscoli CM, Merino M, Brennan TA, Flannery JT, Robboy SJ. Clear cell adenocarcinoma of the vagina and cervix: incidence, undetected disease, and diethylstilbestrol. J Clin Epidemiol 1988;41:593-7.

La Vecchia Cl Franceschi S. Reproductive factors and colorectal cancer. Cancer Causes Control 1991;2:193-200.

Hilakivi-Clarke L, Cho El deAssis S, Olivo S, Ealley El Bouker KB, Welch JN, Khan G, Clarke R, Cabanes A. Maternal and prepubertal diet, mammary developrnent and breast cancer risk. J Nutr 2001 ;1 31 :154S-l57S.

Zava DT and Duwe G. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer l997;27:3l4O.

Hsieh C-Y, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer '(MCF-7) cells in vitro and in vivo. Cancer Res 1998;3833-8. Sathyamoorthy N, Wang TT, Phang JM. Stimulation of pS2 expression by diet-derived compounds. Cancer Res 1994;54:957-61.

Miodini PlFioravanti L, Di Fronzo G, Cappelletti V. The two phyto-oestrogens genistein and quercetin exert different effects on oestrogen receptor function. Br J Cancer 1999;80:1150-5.

Rosenberg Zand RS, Jenkins DJA, Diamandis €P. Agonistic activity of flavonoids and related compounds on steroid hormone receptors. Breast Cancer Res Treat 2000;62:35-49.

Miksicek RJ. Estrogenic flavonoids: structural requirements for biological activity. Proc Soc Exp Biol Med 1995;208:44-50.

Le Bail JC, Varnat F, Nicolas JC, Habrioux G. Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids. Cancer Lett 1998; 13O:209-16.

Breinholt V, Larsen JC. Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCW cell proliferation assay. Chem Res Toxicol 1998;11:622-9.

Gehm BD, McAndrews JM, Chien P-Y, Jameson JL. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc Natl Acad Sci 1997;94:14138-43.

Kuroyanagi M, Arakawa T, Hirayama Y, Hayashi T. Antibacterial and antiandrogen flavonoids from Sophora flavescens. J Natl Prod l999;62:1595- 9.

Rosenberg Zand RS, Jenkins DJ, Diamandis EP. Genistein: a potentiai natural anti-androgen. Clin Chem 2000;46:887-8.

Rosenberg Zand RS, Jenkins DJ, Brown TJ, Diamandis EP. Flavonoids can block PSA production by breast and prostate cancer cell Iines. Clin Chim Acta 2001 (in press).

Davis JN, Muqim N, Bhuiyan Ml Kucuk O, Pienta KJ, Sarkar FH. Inhibition of prostate specific antigen expression by genistein in prostate cancer cells. Int J Oncol 2000;16:1091-7.

Mitchell SH, Zhu W, Young CYF. Resveratrol inhibits the expression and function of the androgen receptor in LNCaP prostate cancer cells. Cancer Res 1999;59:5892-5. Gupta S, Ahmad N, Mohan RR, Husain MM, Mukhtar H. Prostate cancer chemoprevention by green tea: in vitro and in vivo inhibition of testosterone- mediated induction of ornithine carboxylase. Cancer Res 1999;59:2115-20.

Jeong HJ, Shin YG, Kim IH, Peuuto JM. Inhibition of aromatase activity by flavonoids. Arch Pharm Res 1999;22:309-12.

Campbell DR, Kurzer MS. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes. J Steroid Biochern Mol Biol 1993;46:381-8.

Wang Cl Makela T, Hase Tl Adlercreutz Hl Kurzer MS. Lignans and flavonoids inhibit aromatase enzyme in human preadipocytes. J Steroid Biochem Mol Biol 1994;50:205-12.

Le Bail JC, Laroche Tl Marre-Fournier F, Habrioux G. Aromatase and 17beta-hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Lett 1998;133:101-6.

Huang Z, Fasco MJ, Kaminsky LS. Inhibition of estrone sulfatase in human liver microsomes by quercetin and other flavonoids. J Steroid Biochem Mol Bi01 1997;63:9-15.

Schubert W, Eriksson Ur Edgar 8, Cullberg G, Hedner T. Flavonoids in grapefruit juice inhibit the in vitro hepatic metabolism of 17 beta-estradiol. Eur J Drug Metab Pharmacokinet 1995;20:219-24.

Makela SI Poutanen Ml Lehtimaki J, Kostian M-L, Santti R, Vihko R. Estrogen-specific 17$-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1 -62) as a possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 1995;208:51-9.

Ghersevich SA, Poutanen MH, Martikainen HK, Vihko RK. Expression of 17p-hydroxysteroid dehydrogenase in the rat ovary during follicular development and luteinization induced with pregnant mare senirn gonadotrophin and human chorionic gonadotrophin. J Endocrinol 1994;143: 139-50.

Sawetawan C, Milewich L, Word RA, Carr BR, Rainey WE. Compartmentalization of type 1 17p-hydroxysteroid oxidoreductase in the ovary. Mol Cell Endocrinol 1994;99:161-8.

Maentausta O, Boman K, Isomaa V, Stendahl U, Backstrom Tl Vihko R. lmmunohistochemical study of the human 17$-hydroxysteroid and steroid receptors in endometrial carcinoma. Cancer 1992;iO:l551-5. Poutanen M, lsomaa VI Lehto V-PI Vihkro R. lmmunological analysis of 17P- hydroxysteroid in benign and malignant human breast tissue. In J Cancer 1992;50:386-90.

Keung WM. Dietary estrogenic isoflavones are potent inhibitors of beta- hydroxysteroid dehydrogenase of P. testosteronii. Biochem Biophys Res Commun l995;2l 5:1137-44.

Weber KS, Jacobson NA, Setchell KD, Lephart ED. Brain aromatase and Salpha-reductase, regulatory behaviors and testosterone levels in adult rats on phytoestrogen diets. Proc Soc Exp Biol Med l999;22l:l3l-5.

Wang C and Kurzer MS. Phytoestrogen concentration determines effects on DNA synthesis in hurnan breast cancer cells. Nutr Cancer 1997;28:236-47.

Shao ZM, Alpaugh ML, Fontana JA, Barsky SH. Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAFIiCIP1 induction, G2/M arrest, and apoptosis. J Cell Biochem 1998;69:44-54.

Kuntz S, Wenzel U, Daniel H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell iines. Eur J Nutr 1999;38:133-42.

Wenzel U, Kuntz SI Brendel MD, Daniel H. Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. Cancer Res 2000;60:3823-31.

Agullo G, Gamet L, Fernandez Y, Anciaux N, Demigne CI Remesy C. Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinorna cell line (HT29 cells). Cancer Lett 1996:61-70.

Verma SP and Goldin BR. Effect of soy-derived isoflavonoids on the induced growth of MCF-7 cells by estrogenic environmental chemicals. Nutr Cancer 1998;30:232-9.

Weber G, Shen FI Yang H, Prajda NI Li W. Regulation of signal transduction activity in normal and cancer cells. Anticancer Res 1999;19:3703-9.

Agullo G, Gamet-Payrastre LI Manenti S, Viala C, Remesy C, Chap H, Payrastre B. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a cornparison with tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol 1997;53: 1649-57. Weber G, Shen F, Prajda N, Yang H, Li W, Yeh A, Csokay 8, Olah E, Look KY. Regulation of the signal transduction program by drugs. Adv Enzyme Regul 1997;37:35-55.

Scholar EM and Toews ML. Inhibition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein. Cancer Lett 1994;87:159-62.

Schultze-Mosgau MH, Dale IL, Gant TW, Chipman JK, Kerr DJ, Gescher A. Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells. Eur J Cancer 1998;34:1425-31.

Yin F, Giuliano A€, Van Herle AJ. Signal pathways involved in apigenin inhibition of growth and induction of apoptosis of human anaplastic thyroid cancer cells (ARO). Anticancer Res 1999; 19:4297-303.

Kuo ML, Lin JK, Huang TS, Yang NC. Reversion of the transformed phenotypes of v-H-ras NIH3T3 cells by flavonoids through attenuating the content of phosphotyrosine. Cancer Lett 1994;87:91-7.

Peterson G, Barnes S. Genistein inhibits both estrogen and growth factor- stimulated proliferation of human breast cancer cells. Cell Growth Differ 1996;7:1345-5l.

Peterson G, Barnes S. Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation. Prostate 1993;22:335-45.

Kong AN, Yu R, Chen C, Mandlekar S, Primiano T. Signal transduction events elicited by natural products: role of MAPK and caspase pathways in homeostatic response and induction of apoptosis. Arch Pharm Res 2OOO;23: 1-1 6.

Dai RI Jacobson KA, Robinson RC, Friedman FK. Differential effects of flavonoids on testosterone-metabolking cytochrome P450s. Life Sci 1997;61 :PL75-80.

Pagliacci MC, Smacchia MI Migliorati G, Grignani F, Riccardi C, Nicoletti 1. Growth-inhibitory effects of the natural phyto-oestrogen genistein in MCF-7 human breast cancer cells. Eur J Cancer 1994;30:1675-82.

Shen F and Weber G. Synergistic action of quercetin and genistein in human ovarian carcinoma cells. Oncol Res l997;9:597-602. Plaumann 8, Fritsche Ml Rimpler H, Brandner G, Hess RD. Flavonoids activate wild-type p53. Oncogene 1996;13:1605-14.

Shen F, Xue X, Weber G. Tamoxifen and genistein synergistically down- regulate signal transduction and proliferation in estrogen receptor-negative human breast carcinoma MDA-ME-435 cells. Anticancer Res 1999;19:16S7- 62.

Segaert S, Courtois S, Garmyn M, Degreef H, Bouillon R. The flavonoid apigenin suppresses vitamin D receptor expression and vitamin D responsiveness in normal human keratinocytes. Biochem Biophys Res Commun 2000;268:237-41.

So FV, Guthrie N, Chambers AF, Carroll KK. Inhibition of proliferation of estrogen receptor-positive MCF-7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen. Cancer Lett l997;ll2:l27- 33.

Wang TT, Phang JM. Effects of estrogen on apoptotic pathways in human breast cancer cell line MCF-7. Cancer Res 1995;55:2487-9.

Makela S, Davis VL, Tally WC, Korkman J, Salo L, Vihko R, Santii Fi, Korach KS. Dietary estrogens act through estrogen receptor mediated processes and show no antiestrogenicity in cultured breast cancer cells. Env health Persp 1994; 102:135-49.

Santell RC, Kieu N, Helferich WG. Genistein inhibits growth of estrogen- independent human breast cancer cells in culture but not in athymic mice. J Nutr 2000; t 3O:l665-9.

Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med 1997;22:749-60.

Chan T, Galati G, O'Brien PJ. Oxygen activation during peroxidase catalysed metabolism of flavones or flavanones. Chem Eiol lnteract 1999;122:15-25.

Galati G, Chan T, Wu 0, O'Brien PJ. Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalyzed redox cycling of flavonoids. Chem Res Toxicol 1999;12:52l-S.

Ruiz-Larrea MB, Mohan AR, Paganga G, Miller NJ, Bolweil GP, Rice-Evans CA. Antioxidant activity of phytoestrogenic isoflavones. Free Radic Res 1997;26:63-70.

Johnson MK and Loo G. Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. Mutat Res 2000;459:211-8. Mitchell JH, Gardner PT, McPhail DE, Morrice PC, Collins AR, Duthie GG. Antioxidant efficacy of phytoestrogens in chemical and biological model systems. Arch Biochem Biophys 1998;360:142-8.

Arora A, Nair MG, Strasburg GM. Antioxidant activities of isoflavones and their biological metabolites in a liposomal system. Arch Biochem Biophys 1998;356:133-41.

Jha JC, von Recklinghausen G, Zilliken F. Inhibition of in vitro microsomal lipid peroxidation by isoflavonoids. Biochem Pharmacol 1985;34:1367-9.

Wei H, Bowen R, Cai Q, Barnes S, Wang Y. Antioxidant and antipromotional effects of the soybean isoflavone genistein. Proc Soc Exp Biol Med 1995;208:124-30.

Breinholt V, Laundsen ST, Dragsted LO. Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat. Xenobiotica 1999;29:1227-40.

Huber WW, McDaniel LP, Kaderlik KR, Teitel CH, Lang NP, Kadlubar FF. Chemoprotection against the formation of colon DNA adducts from the food- borne carcinogen 2-amino-1-methyl-6-phenyIimidazo[4,5b]pyridine (PhlP) in the rat. Mutat Res 1997;376:115-22.

Duthie GG. Determination of activity of antioxidants in human subjects. Proc Nutr Soc l999;S8:lOlS-24.

Halliwell B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Radic Res 1996;25:57-74.

Troll W, Wiesner R, Shellabarger CJ, Holtzman SI Stone JP. Soybean diet lowers breast tumor incidence in irradiated rats. Carcinogenesis f98O;l:469- 72.

Ohta Tl Nakatsugi S, Watanabe K, Kawamon Tl lshikawa FI Morotomi M, Sugie S, Toda T, Sugimura Tl Wakabayashi K. lnhibitory effects of Bifidobacterium-ferrnented soy milk on 2-amino-1-methyl-6- phenylimidazo[4,5-blpyridine-induced rat mammary carcinogenesis, with a partial contribution of its comportent isoflavones. Carcinogenesis 2000;21:937-41.

Constantinou Al, Krygier AE, Mehta RR. Genistein induces maturation of cultured human breast cancer cells and prevents tumor growth in nude mice. Am J Clin Nutr 1998;68:1426S-30s. Appelt LC and Reicks MM. Soy induces phase II enzymes but does not inhibit dimethylbenz[a]anthracene-induced carcinogenesis in female rats. J Nutr 1999;129:1820-6.

Cohen LA, Zhao Z, Pittman B, Scimeca JA. Effect of intact and isoflavone- depleted soy protein on NMU-induced rat mammary tumorigenesis. Carcinogenesis 2000;21:929-35.

Fritz WA, Coward L, Wang J, Lamartiniere CA. Dietary genistein: perinatal mammary cancer prevention, hioavailability and toxicity testing in the rat. Carcinogenesis 1998;19:2151-8.

Lamartiniere CA, Moore JB, Brown NM, Thompson R, Hardin MJ, Barnes S. Genistein suppresses mammary cancer in rats. Carcinogenesis 1995; 16:2833-40.

Murrill WB, Brown NM, Zhang JX, Manolillo PA, Barnes S, Lamartiniere CA. Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 1996; 17:14Sl-7.

Gotoh TlYamada K, Yin HlIto A, Kataoka Tl Dohi K. Chemoprevention of N- nitroso-N-methylurea-induced rat mammary carcinogenesis by soy foods or biochanin A. Jpn J Cancer Res 1998;89:137-42.

Kato K, Takahashi S, Cui L, Toda Tl Suzuki S, Futakuchi Ml Sugiura S, Shirai T. Suppressive effects of dietary genistin and daidzin on rat prostate carcinogenesis. Jpn J Cancer Res 2000;91:786-91.

Thiagarajan DG, Bennink MR, Bourquin LD, Kavas FA. Prevention of precancerous colonic lesions in rats by soy flakes, soy flour, genistein, and calcium. Am J Clin Nutr l998;68:13948-98.

Deschner EE, Ruperto J, Wong G, Newmark HL. Quercetin and rutin as inhibitors of azoxymethanol-induced colonic neoplasia. Carcinogenesis 1991 ;12:1193-6.

Oeschner EE, Ruperto JF, Wong GY, Newmark HL. The effect of dietary quercetin and rutin on AOM-induced acute colonic epithelial abnonnalities in mice fed a high-rat diet. Nutr Cancer 1993;20:199-204.

Yang K, Lamprecht SA, Liu Y, Shinozaki H, Fan K, Leung Dl Newmark H, Steele VE, Kelloff GJ, Lipkin M. Chemoprevention studies of the flavonoids quercetin and rutin in normal and azoxymethane-treated mouse colon. Carcinogenesis 2000;21:1655-60. Tanaka Tl Makita H, Kawabata K, Mon Hl Kakumoto M, Satoh KI Hara A, Sumida Tl Tanaka Tl Ogawa H. Chernoprevention of azoxyrnethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosrnin and hesperidin. Carcinogenesis 1997; l8:957-65.

Tanaka Tl Kawabata K, Kakumoto Ml Hara A, Murakami A, Kuki W, Takahashi Y, Yonei Hl Maeda Ml Ota Tl Odashima SI Yamane T, Koshimizu K, Ohigashi H. Citnis auraptene exerts dose-dependent chemopreventive activity in rat large bowel tumorigenesis: the inhibition correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes. Cancer Res 1998;58:2550-6.

Miyagi Y, Om AS, Chee KM, Bennink MR. lnhibition of azoxymethane- induced colon cancer by orange juice. Nutr Cancer 2000;36:224-9.

Mutoh Ml Takahashi M, Fukuda K, Komatsu H, Enya Tl Matsushima-Hibiya Y, Mutoh H, Sugimura Tl Wakabayashi K. Suppression by flavonoids of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells: structure-activity relationship. Jpn J Cancer Res 20001;91:686-91.

Liang YC, Huang YT, Tsai SH, Lin-Shiau SY, Chen CF, Lin JK. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis 1999;20:194S-52.

Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN. Modulation of apoptosis and Bcl-2 expression by prostaglandins E2 in human colon cancer cells. Cancer Res 1998;58:362-6.

Hao XI Bishop AE, Wallace Ml Wang H, Willcocks TC, Maclouf J, Polak JM, Knight S, Talbot IC. Early expression of cyclooxygenase-2 during sporadic colorectal carcinogenesis. J Pathol 1999;187:295-301.

Elder DJ and Paraskeva C. COX-2 inhibitors for colorectal cancer. Natl Med 1998;4:392-3.

Chae YH, Marcus CB, Ho DK, Cassady JM, Baird WM. Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared frorn rats treated with cytochrome P-450 inducers. Cancer Lett 1991;60:15-24.

Shertzer HG, Puga A, Chang Cl Smith Pl Nebert DW, Setchell KD, Dalton TP. Inhibition of CYP1A1 enzyme activity in rnouse hepatoma cell culture by soybean isoflavones. Chem Biol lnteract 1999; 123:31-49. Maskarinec G, Singh SI Meng L, Franke AA. Dietary soy intake and urinary isoflavone excretion among women from a multiethnic population. Cancer Epidemiol Biomarkers Prev 1998;7:613-9.

Cheng Z, Zheng W, Custer LJ, Dai Q, Shu XO, Jin F, Franke AA. Usual dietary consumption of soy fmds and its correlation with the excretion rate of isoflavonoids in overnight urine sarnples among Chinese women in Shanghai. Nutr Cancer 1999;33:82-7.

Adlercreutz Hl Honjo H, tligashi A, Fotsis TI Hamalainen El Hasegawa Tl Okada H. Urinary excretion of Iignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr 1991;54:1093-100.

Karr SC, Lampe JW, Hutchins AM, Slavin JL. Urinary isoflavonoid excretion in humans is dose dependent at low to moderate levels of soy-protein consumption. Am J Clin Nutr 1997;66:46-51.

Cassidy A, Bingham S, Setchell KD. 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.

Lu LJ, Anderson KE, Grady JJ, Kohen FI Nagamani M. Decreased ovarian hormones during a soya diet: implications for breast cancer prevention. Cancer Fies 2000;60:4112-21.

Duncan AM, Merz BE, Xu X, Nagel TC, Phipps WR, Kurzer MS. Soy isoflavones exert modest honnonal effects in premenopausal women. J Clin Endocrinol Metab l999;84:192-7.

Hargreaves DF, Potten CS, Harding Cl Shaw LE, Morton MS, Roberts SA, Howell A, Bundred NJ. Two-week dietary soy supplementation has an estrogenic effect on normal prernenopausal breast. J Clin Endocrinol Metab 1999;84:4017-24.

Martini MC, Dancisak BB, Haggans CJ, Thomas W, Slavin JL. Effects of soy intake on sex hormone metabolism in premenopausal women. Nutr Cancer 1999;34:133-9.

Lu W, Anderson KE, Grady JJ, Nagamani M. Effects of soya consumption for one month on steroid hormones in premenopausal women: implications for breast cancer risk reduction. Cancer Epidemiol Biomarkers Prev 1996;5:63- 70.

Fortunati N. Sex hormone-binding globulin: not only a transport protein. What news is around the corner? J Endocrinol lnvest 1999;22:223-34. Adlercreutz Hl Mousavi Y, Clark J, Hockerstedt K, Hamalainen El Wahala K, Makela Tl Hase T. Dietary phytoestrogens and cancer: in vitro and in vivo studies. J Steroid Biochem Mol Biol 1992;41:331-7.

Duncan AM, Merz-Demlow BE, Xu X, Phipps WR, Kurzer MS. Premenopausal equol excretors show plasma hormone profiles associated with lowered risk of breast cancer. Cancer Epiderniol Biomarkers Prev 2OOO;9:581-6.

Lu LJ, Cree M, Josyula S, Nagamani Ml Grady JJ, Anderson KE. lncreased urinary excretion of 2-hydroxyestrone but not 16a-hydroxyestrone in premenopausal women during a soya diet containing isoflavones. Cancer Res 2OOO;6O: 1299-305.

Wu AH, Stanczyk FZ, Hendrich S, Murphy PA, Zhang Cl Wan Pl Pike MC. Effects of soy foods on ovanan function in premenopausal women. Br J Cancer 2OOO;82: 1879-86.

Nagata Cl Takatsuka N, Inaba S, Kawakami N, Shimizu H. Effects of soymilk consumption on serum estrogen concentrations in premenopausal Japanese women. J Natl Cancer lnst 1998;90:1830-5.

Petrakis NL, Barnes S, King EB, Lowenstein JI Wiencke J, Lee MM, Miike R, Kirk M, Coward L. Stimulatory influence of soy protein isolate on breast secretion in pre- and postrnenopausal women. Cancer Epidemiol Biomarkers Prev 1996;5:785-94.

McMichael-Phillips DF, Harding Cl Morton M, Roberts SA, Howell A, Potten CS, Bundred NJ. Effects of soy-protein supplementation on epithelial proliferation in the histologically normal human breast. Am J Clin Nutr l998;68: 1431S-5s.

Jenkins DJ, Kendall CW, Garsetti Ml Rosenberg Zand RS, Jackson CJ, Agarwal S, Rao AV, Diamandis EP, Parker Tl Faulkner Dl Vuksan V, Vidgen E. Effect of soy protein foods on low-density lipoprotein oxidation and ex vivo sex hormone receptor actnrity - a controlled crossover trial. Metabolism 2000;49:537-43.

Labrie F, Luu-The V, Lin SX, Simard J, Labrie Cl EI-Alfy Ml Pelletier G, Belanger A. Intracrinology: role of the family of l'la-hydroxysteroid dehydrogenases in human physiology and disease.

Klimar MV and Tindall DJ. Transcriptionai regulation of the steroid receptor genes. Prog Nucl Acid Res Mol Bi01 1998;59:289-306. Robyr D, Wolffe AP, Wahli W. Nuclear hormone receptor coregulators in action: diversity for shared tasks. Mol EndocrinoI 2000; l4:329-47.

Q'Malley 8. The steroid receptor superfamily: More excitement predicted for the future. Mol Endocrinol 1990;4:363-9.

Parker MG. Structure and function of nuclear hormone receptors. Seminars Cancer Biol 1990; 1:81-7.

Laudet VI Hanni ClColl J, Catzeflis F, Stehelin O. Evolution of the nuclear receptorgene superfamily. EMBO J 1992;11:1003-13.

Landers JP and Spelsberg TC. New concepts in steroid hormone action: Transcription factors, proto-oncogenes, and the cascade model for steroid regulation of gene expression. Crit Rev Eukaryot Gene Exp 1992;2:19-63.

Lindzey J, Kurnar MV, Grossman Ml Young C, Tindall DJ. Molecular mechanisms of androgen action. Vitarnins Hormones 1994;49:383-432.

Tsai M-J and OIMatley BW. Molecular mechanisms of action of steroidhhyroid receptor superfamily. Annu Rev Biochern l994;63:45 1-86.

O'Malley BW, Schrader WT, Mani S, Smith C, Weigel NL. Conneely OM, Clark JH. An alternative ligand-independent pathway for activation of steroid receptors. Recent Prog Hormone Res 1995;50:333-47.

Barettino D, Vivanco Ruiz MM, Stunnenberg HG. Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor. EMBO J 1994; 1313039-49.

Danielian PS, White R, Lees JA, Parker MG. Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 1992; 11 : 1025-33.

Durand B, Saunders M, Gaudon ClRoy 6,Losson R, Chambon P. Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: Presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J 1994;13:5370-82.

Saatciogh FI Bartunek P, Deng T, Zenke M, Karin M. A conserved C- terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor). Mol CeIl Bi01 l993;13:3675-85. Segnitz B and Gehring U. Subunit structure of the nonactivated human estrogen receptor. Proc Natl Acad Sci USA 1995;92:2179-83.

Kuiper GG and Brinkmann AO. Steroid hormone receptor phosphorylation: Is there a physiological role? Mol Cell Endocrinol 1994;100:103-7.

White R and Parker MG. Molecuiar mechanisms of steroid hormone action. Endocrine-Related Cancer l998;5:1-14.

Bocquel MT, Kumar V, Stricker Cl Chambon Pl Gronemeyer H. The contribution of the N- and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell-specific. Nucleic Acids Res 1989; 17:258 1-95.

Meyer ME, Gronemeyer H, Turcotte BI Bocquel MT, Tasset D, Chambon P. Steroid hormone receptors compete for factors that mediate their enhancer function. Cell 1989;57:433-42.

Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M. Estrogsn receptor-association proteins: Possible mediators of hormone- induced transcription. Science 1994;264:1455-8.

Horowitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L. Nuclear receptor coactivators and corepressors. Mol Endocrinol 1996;10:1167-77.

Glass CK, Rose DW, Rosenfeld MG. Nuclear receptor coactivators. Curr Opin Cell Bi01 1997;9:222-32.

Torchia J, Glass Cl Rosenfeld MG. Co-activators and corepressors in the integration of transcriptional responses. Curr Opin Cell Biol 1998;10:373-83.

Pujol Pl Rey JM, Nirde P, Roger Pl Gastaldi M, Laffargue F, Rochefort H, Maudelonde T. Differential expression of estrogen receptor-alpha and -beta messenger RNAs as a potential market of ovarian carcinogenesis. Cancer Res 1998;58:5367-73.

Kuiper GG, Lemmen JG, Carlsson 6,Corton JC, Safe SH, van der Saag PT, van der Burg 6, Gustafsson JA. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998;139:4252- 63.

Kida N, Yoshimura T, Mori K, Hayashi K. Hormonal regulation of synthesis and secretion of pS2 protein relevant to growth of human breast cancer cells (MCF-7). Cancer Fies 1989;49:3494-8. Masiakowski Pl Breathnach RI Bloch JI Gannon FI Krust A, Chambon P. . Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human breast cancer cell line. Nucleic Acids Res 1982;10:7895-903.

Plaut AG. Trefoil peptides in the defense of the gastrointestinal tract. N Engl J Med 1997;336:506-7.

Suemori SI Lynch-Devaney K, Podolsky DK. Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific mernber of the trefoil protein family. Proc Natl Acad Sci U S A 1991;88:11017-21.

Rio MC, Bellocq JP, Daniel JY, Tomasetto C, Lathe RI Chenard MP, Batzenschlager A, Chambon P. Breast cancer-associated pS2 protein: synthesis and secretion by normal stomach mucosa. Science 1988;241:705- 8.

Hanby AM, Poulsom RI Singh S, Elia G, Jeffery RE, Wright NA. Spasmolytic polypeptide is a major antrat peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach. Gastroenterology 1993;l 05:1110-6.

Piggott NH, Henry JA, May FE, Westley BR. Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry. J Pathol 1991;163:95-104.

Dignass A, Lynch-Devaney KI Kindon H, Thim LI Podolsky DK. Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway. J Clin lnvest 1994;94:376-83.

Williams R, Stamp GW, Gilbert C, Pignatelli M, Lalani EN. pS2 transfection of rnurine adenocarcinoma cell line 41 0.4 enhances dispersed growth pattern in a 3-D collagen gel. J Cell Sci 1996;109:63-71.

Playford RJ, Marchbank T, Calnan DP, Calam JI Royston P, Batten JJ, Hansen HF. Epidermal growth factor is digested to smaller, less active forms in acidic gastric juice. Gastroenterology 1995;108:92-101.

May FE, Westley BR. Trefoil proteins: their role in normal and malignant ce1ls.J Pathol 1997;t 83:4-7.

Wright NA, Poulsom R, Stamp GW, Hall PA, Jeffery RE, Longcroft JM, Rio MC, Tomasetto C, Chambon P. Epidemal growth factor (EGWURO) induces expression of regulatory peptides in damaged human gastrointestinal tissues. J Pathol 1990; l62:279-84. Foekens JA, Rio MC, Seguin Pl van Putten WL, Fauque JI Nap Ml Klijn JG, Chambon P. Prediction of relapse and suwival in breast cancer patients by pS2 protein status. Cancer Fies 1990;50:3832-7.

Rio MC, Bellocq JP, Gairard BI Rasmussen UB, Krust A, Koehi Cl Calderoli H, Schiff V, Renaud R, Chambon P. Specific expression of the pS2 gene in subclasses of breast cancers in comparison with expression of the estrogen and progesterone receptors and the oncogene ERBB2. Proc Natl Acad Sci U S A 1987;84:9243-7.

Ju YH, Carlson KE, Sun J, Pathak Dl Katzenellenbogen ES, Katzenellenbogen JA, Helferich WG. Estrogenic effects of extracts from cabbage, ferrnented cabbage, and acidified brussels sprouts on growth and gene expression of estrogen-dependent human breast cancer (MCF-7) cells. J Agric Food Chem 2000;48:4628-34.

Jansen RL, Hupperets PS, Arends JW, Joosten-Achjanie SR, Volovics A, Hillen HF, Schouten HC. pS2 is an independent prognostic factor for post- relapse survival in pnmary breast cancer. Anticancer Res 1998; 18:577-82.

Diamandis EP. lmmunoassays with time-resolved fluorescence spectroscopy: principles and applications. Clin Biochem 1988;21:139-50.

Christopoulos TK, Diamandis EP. Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates. Anal C hem 1992;64:342- 6.

Diamandis EP. Multiple labeling and time-resolvable fluorophores. Clin Chem 1991;37:1486-91.

Ferguson RA, Yu Hl Kalyvas M, Zammit SI Diamandis EP. Ultrasensitive detection of prostate-specific antigen by a time-resolved immunofluorometnc assay and the lmmulite immunochemiluminescent third-generation assay: potential applications in prostate and breast cancers. Clin Chem 1996;42:675- 84.

Diamandis EP, Christopoulos TK, Khosravi MJ. Development of in-house immunological assays. In: Diamandis EP, Christopoulos TK, eds. Immunoassay. San Diego: Academic Press 1996:555-68.

May FE, Westley BR. Expression of human intestinal trefoil factor in malignant cells and its regulation by oestrogen in breast cancer ce1ls.J Pathol 1997;182:4O4-13. Soubeyran 1, Quenel NICoindre JM, Bonichon FI Durand Ml Wafflart J, Mauriac L. pS2 protein: a marker improving prediction of response to neoadjuvant tamoxifen in post-menopausal breast cancer patients. Br J Cancer l996;74:ll2O-5.

Foekens JA, van Putten WL, Portengen HI de Koning HY, Thirion BI Alexieva-Figusch JI Klijn JG. Prognostic value of PS2 and cathepsin D in 710 human primary breast tumors: multivariate analysis. J Clin Oncol 1993;11:899-908.

Gion M, Mione R, Pappagallo GL, Gatti CI Nascimben O, Bari M, Leon AE, Vinante O, Bruscagnin G. PS2 in breast cancer-alternative or complementary tool to steroid receptor status? Evaluation of 446 cases. Br J Cancer 1993;68:374-9.

Foekens JA, Portengen HI Look MP, van Putten WL, Thirion BI Bontenbal Ml Klijn JG. Relationship of PS2 with response to tamoxifen therapy in patients with recurrent breast cancer. Br J Cancer l994;7O:l2l7-23.

Zarghami N, Grass LI Diamandis €P. Steroid hormone regulation of prostate- specific antigen gene expression in breast cancer. Br J Cancer 1997;75:579- 88.

Howell A, DeFriend Dl Robertson J, Blarney R, Walton P. Response to a specific antioestrogen (ICI 182780) in tamoxifen-resistant breast cancer. Lancet 1995;345:29-30.

Wakeling AE, Dukes Ml Bowler J. A potent specific pure antiestrogen with clinical potential* Cancer Res 1991;51:3867-73.

Fawell SE, White R, Hoare S, Sydenham MI Page Ml Parker MG. Inhibition of estrogen receptor-DNA binding by the "pure" antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerkation. Proc Nati Acad Sci U S A 1990;87:6883-7.

Gibson MK, Nemrners LA, Beckman WC Jr, Davis VL, Curtis SW, Korach KS. The mechanism of ICI 164,384 antiestrogenicity involves rapid loss of estrogen receptor in uterine tissue. Endocrinology 1991;129:2OOO-lO.

Dauvois S, White R, Parker MG. The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci 1993;l O6:137%88.

Howell A, Downey S, Anderson E. New endocrine therapies for breast cancer. Eur J Cancer 1996;32A:576-88. Kangas L, Nieminen AL, Blanco G, Gronroos MlKallio S, Karjalainen A, Perila Ml Sodervail Ml Toivola R. A new triphenyiethylene compound, Fc- 1157a. II. Antitumor effects. Cancer Chemother Pharmacol l986;l7:lO9-l3.

Favoni RE, de Cupis A. Steroidal and nonsteroidal oestrogen antagonists in breast cancer: basic and clinical appraisal. Trends in Pharmaceutical Sciences l998;I9:406-15.

Loser R, Seibel KI Roos W, Eppenberger U. In vivo and in vitro antiestrogenic action of 3-hydroxytamoxifen, tamoxifen and 4-hydroxytamoxifen. Eur J Cancer Clin Oncoî 1985;21:985-90.

Black LJ, Jones CD, Falcone JF. Antagonism of estrogen action with a new benzothiophene derived antiestrogen. Life Sci l983;32:1031-6.

Toko Tl Sugimoto Y, Matsuo KI Yamasaki R, Takeda S, Wierzba K, Asao T, Yamada Y. TAT-59, a new triphenylethylene derivative with antitumor activity against hormone-dependent tumors. Eur J Cancer 1990;26:397-404.

Chander SK, McCague R, Luqmani Y, Newton Cl Dowsett M, Jarman M, Coombes RC. Pyrrolidino-4-iodotamoxifen and 4-iodotamoxifen, new analogues of the antiestrogen tamoxifen for the treatrnent of breast cancer. Cancer Res 1991;51:5851-8.

Kraus WL, Weis KE, Katzenellenbogen BS. lnhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist- and antagonist-occupied progestin receptors. Mol Cell Biol 1995;15:1847-57.

Jeng MH, Langan-Fahey SM, Jordan VC. Estrogenic actions of RU486 in hormone-responsive MCF-7 human breast cancer cells. Endocnnology 1993; 132:2622-30,

Nawaz Z, Stancel GM, Hyder SM. The pure antiestrogen ICI 182,780 inhibits progestin-induced transcription. Cancer Res 1999;59:372-6.

Catherino WH, Jordan VC. lncreasing the number of tandem estrogen response elements increases the estrogenic activity of a tamoxifen analogue. Cancer Leît 1995;92:39-47.

Hall RE, Tilley WD, McPhaul MJ, Sutherland RL. Regulation of androgen receptor gene expression by steroids and retinoic acid in human breast- cancer cells. Int J Cancer t992;52:778-84. 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 Res Treat 1994;32:291-300.

Black MH, Magklara A, Obiezu CV, Melegos DN, Diamandis EP. Development of an ultrasensitive immunoassay for human glandular kallikrein (hK2) with no cross reactivity from prostate specific antigen. In Press.

Phillips A, Demarest K, Hahn DW, Wong F, McGuire JL. Progestational and androgenic receptor binding affinities and in vivo activities of norgestirnate and other progestins. Contraception 1990;41:399-410.

Lange CA, Richer JK, Shen T, Horowitz KB. Convergence of progesterone and epidermal growth factor signaling in breast cancer. Potentiation of rnitogen-activated protein kinase pa?hways. J Biol Chem 1998;273:3l308-16.

Richer JK, Lange CA, Manning NG, Owen G, Powell R, Horowitz KB. Convergence of progesterone with growth factor and cytokine signaling in breast cancer. Progesterone receptors regulate signal transducers and activators of transcription expression and activity. J Biol Chem 1998;273:31317-26.

Weaver CA, Springer PA, Katzenellenbogen BS. Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2- chloramphenicol acetyl transferase fusion genes transfected into MCF-7 human breast cancer cells. Mol Endocrinol 1988;2:936-45.

Xu X, Duncan AM, Men BE, Kurzer MS. Effects of soy isoflavones on estrogen and phytoestrogen metabolism in premenopausal women. Cancer Epidemiol Biomarkers Prev 1998;7:11Ol-8.

Dardes RC, Jordan VC. Novel agents to modulate oestrogen action. Br Med Bull 2000;56:773-86.

Delmas PD. Clinical use of selective estrogen receptor modulators. Bone l999;25:115-8

Weryha G, Pascal-Vigneron V, Klein M, Leclere J. Selective estrogen receptor modulators. Curr Opin Rheumatol 1999;11:301-6.

Ansbacher R. Selective estrogen receptor modulators. Am J Obstet Gynecol 1999; 181 :lO36.

Hochner-Celnikier D. Pharmacokinetics of raloxifene and its clinical applications. Eur J Obstet Gynecol Reprod Biol 1999;85:23-9. Brzezinski A, Debi A. Phytoestrogens: the "natural" selective estrogen receptor modulators? Eur J Obstet Gynecol Reprod Biol 1999;85:47-51.

Rimm EB, Katan MB, Ascherio A, Stampfer MJ, Willett WC. Relation between intake of flavonoids and risk for coronary heart disease in male health professionals. Ann lntern Med l996;125:384-9.

Noroozi M, Angerson WJ, Lean ME. Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. Am J Clin Nutr 1998;67:1210- 8.

Kuzumaki Tl Kobayashi Tl lshikawa K. Genistein induces p21(Cipl/WAFl) expression and blocks the G1 to S phase transition in mouse fibroblast and melanoma cells. Biochem Biophys Res Commun 1998;2Sl :29l -5.

Constantinou Al, Kamath NIMurley JS. Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle, and induces apoptosis of human breast adenocarcinorna MCF-7 cells. Eur J Cancer 1998;34:1927-34.

Fotsis T, Pepper MS, Aktas E, Breit S, Rasku S, Adlercreutz Hl Wahala KI Montesano RI Schweigerer L. Flavonoids, dietav-derived inhibitors of ce11 proliferation and in vitro angiogenesis. Cancer Res 1997;57:2916-21.

Hempstock J, Kavanagh JP, George NJ. Growth inhibition of prostate ceil lines in vitro by phyto-oestrogens. Br J Urol 1998;82:560-3.

Fioravanti L, Cappelletti VI Miodini Pl Ronchi E, Brivio M, Di Fronzo G. Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro. Cancer Lett 1998;130:143-52.

Choi YH, Zhang L, Lee WH, Park KY. Genistein-induced G2/M arrest is associated with the inhibition of cyclin 01 and the induction of p21 in human breast carcinoma cells. Int J Oncol 1998;l -6.

Miksicek RJ. Commonly occurring plant flavonoids have estrogenic activity. Mol Pharmacol 1993;44:37-43.

Rosenberg RS, Grass LI Jenkins DJ, Kendall CW, Diamandis EP. Modulation of androgen and progesterone receptors by phytochemicals in breast cancer cell lines. Biochem Biophys Res Commun 1998;248:935-9.

Kitaoka M, Kadokawa Hl Sugano M, Ichikawa K, Taki Ml Takaishi S, lijima Y, Tsutsumi S, Boriboon M, Akiyama T. Prenylflavonoids: a new class of non- steroidal phytoestrogen (Part 1). Isolation of 8-isopentenylnaringenin and an initial study on its structure-activity relationship. Planta Med 1998;64:511-5.

Hall RE, Lee CSL, Alexander IE, Shine J, Clarke CL, Sutherland RL. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorîicoid receptor messenger RNA levels. Int J Cancer 1WO;46:lO8l-7.

Rosenberg Zand RS, Jenkins DJ, Diamandis EP. Developrnent and evaluation of a cornpetitive time-resolved immunofluorometric assay for the estrogen-regulated protein pS2. J Clin Lab Anal 1999; l3:241-5.

Cleutjens KB, van der Korput HA, van Eekelen CC, van Rooij HC, Faber PW, Trapman J. An androgen response element in a far upstrearn enhancer region is essential for high, androgen-regulated activity of the prostate- specific antigen promoter. Mol Endocrinol 1997; 11 : 148-61.

Giugliano D. Dietary antioxidants for cardiovascular prevention, Nutr Metab Cardiovasc Dis 2000;10:38-44.

Yochum C, Kushi LH, Meyer K, Folsom AR. Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. Am J Epidemiol 1999;149:943-9.

Garcia-Closas RI Gonzalez CA, Agudo A, RiboIi E. lntake of specific carotenuids and flavonoids and the risk of gastric cancer in Spain. Cancer Causes Control 1999;t 0:71-5.

Hughes DA. Effects of dietary antioxidants on the immune function of rniddte- aged aduits. Proc Nutr Soc 1999;58:79-84.

Hassig A, Liang WX, Schwabl H, Stampfli K. Flavonoids and tannins: plant- based antioxidants with vitamin character. Med Hypotheses 1999;52:479-81.

Critchfield JW, Butera ST, Folks TM. Inhibition of HIV activation in latently infected cells by flavonoid compounds. AlDS Res Hum Retroviruses l996;12:39-46.

Nestel PJ, Pomeroy S, Kay SIKomesaroff Pl Behrsing J, Cameron JD, West L. lsoflavones frorn red cIover improve systemic artenal compliance but not plasma lipids in menopausal wornen. J Clin Endocrinol Metab 1999;84:895-8.

Albertazzi P, Pansini FI Bonaccorsi G, Zanotti L, Forini E, De Aloysio O. The effect of dietary soy supplementation on hot flushes. Obstet Gynecol 1998;91:6-11. Fauconneau 8, Waffo-Teguo Pl Huguet FIBarrier LIDecendit A, Metillon JM. Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci 1997;61:2103-10.

Lean ME, Norooti MI Kelly 1, Burns J, Talwar DI Sattar N, Crozier A. Dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA. Diabetes 1999;48:176-81.

Lavy A, Fuhrman B, Markel A, Dankner G, Ben-Amotz A, Presser Dl Aviram M. Effect of dietary supplementation of red or white wine on human blood chemistry, hematology and coagulation: favorable effect of red wine on plasma high-density lipoprotein. Ann Nutr Metab 1994;38:287-94.

Tiwari RK, Geliebter J, Garikapaty VP, Yedavelli SPI Chen SI Mittelman A. Anti-tumor effects of PC-SPES, an herbal formulation in prostate cancer. Int J Oncol 1999; l4:713-9.

DiPaola RS, Zhang HI Lambert GH, Meeker R, Licitra El Rafi MM, Zhu BT, Spaulding Hl Goodin SI Toledano MB, Hait WN, Gallo MA. Clinical and biologic activity of an estrogenic herbal combination (PC-SPES) in prostate cancer. N EngI J Med 1998;339:785-91.

Xu LI Glass CK, Rosenfeld MG. Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Gen Dev 1999;g:l 40-7.

Lien EJ, Ren S, Bui HH, Wang R. Quantitative structure-activity relationship analysis of phenolic antioxidants. Free Radic Biol Med 1999;26:285-94.

Zheng W, Dai Q, Custer LJ, Shu XO, Wen WQ, Jin F, Franke AA. Urinary excretion of isoflavonoids and the risk of breast cancer. Cancer Epiderniol Biomarkers Prev 1999;8:35-40. O'Shaughnessy JA. Chemoprevention of breast cancer. JAMA 1996;275: 1349-53.

Jacobsen BK, Knutsen SF, Fraser GE. Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study. Cancer Causes Control 1998;9:553-7. Shao ZM,Wu J, Shen U,Barsky SH. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 1998;58:4851-7. Franke AA, Cooney RV, Custer LJ, Mordan LJ, Tanaka Y. Inhibition of neoplastic transformation and bioavailability of dietary flavonoid agents. Adv Exp Med Biol 1998;439:237-48. Anthony MS, Clarkson TB, Williams JK. Effects of soy isoflavones on atherosclerosis: potential mechanisms. Am J Clin Nutr l998;68: 1390s- 1393s. Kerry N, Abbey M. The isoflavone genistein inhibits copper and peroxyl radical mediated low density lipoprotein oxidation in vitro. Atherosclerosis 1998; 140:341-7. lshimi Y, Miyaura Ci Ohmura M, Onoe Y, Sato TlUchiyama Y, Ito Ml Wang X, Suda T, lkegami S. Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency. Endocrinology 1999;140:1893-900.

Mercer SL, Goel V, LevylG, Ashbury FD, lverson OC, lscoe NA. Prostate cancer screening in the midst of controversy: Canadian men's knowledge, beliefs, utilization and futther intentions. Canad J Pub Heaith 1997;88:327- 32.

Potosky AL, Miller BA, Albertsen PC, Krarner BS. The role of increasing detection in the rising incidence of prostate cancer. JAMA 1995;273:548-52.

Krongrad A, Lai S, Vidal EM. The significance of changing trends in prostate cancer incidence and mottality. Semin Urol Oncol 1998; l6:30-4.

Lefevre ML. Prostate cancer screening: more harm than good? Am Fam Physician 1998;58:432-8.

Villers A, Soulie M, Hailloto, Boccon-Gibod L. Prostate cancer screening (Ill): risk factors, natural history, course without treatment. Characteristics of detected cancers. Prog Urol l997;7:655-6l.

Hayes RB, Ziegler RG, Gridley G, Swanson C, Greenberg RS, Swanson GM, Schoenberg JB, Silverrnan DT, Brown LM, Pottern LM, Lii J, Schwartz AG, Fraumeni JF Jr, Hoover RN. Dietary factors and risks for prostate cancer among blacks and whites in the United States. Cancer Epidemiol Biomarkers Prev 1999;8:25-34.

Fradet Y, Meyer F, Bairati 1, Shadmani R, Moore L. Dietary fat and prostate cancer progression and survival. Eur Urol 1999;35:388-91. Cerhan JR, Torner JC, Lynch CF, Rubenstein LM, Lemke JH, Cohen MB, Lubaroff DM, Wallace RB. Association of smoking, body mass, and physical activity with risk of prostate cancer in the Iowa 65+ Rural Health Study (United States). Cancer Causes Control 1997;8:229-38.

Labrie FI DupondA, Belanger A, Cusan L, Lacourciere Y, Monfette G, Laberge JG, Emond JP, Fazekas AT, Raynaud JP, Husson JM. New hormonal therapy with an LH-RH agonist and antiandrogen. Clin Invest Med 1982;5:267-75.

Crawford ED, Eisenberger MA, McLeod DG, Spaulding JT, Benson R, Dorr FA, Blumenstein BA, Davis MA, Goodman PJ. A controlled trial of leuproiide with and without flutamide in prostatic carcinoma. N Engl J Med 1989;321:419-24.

Auclerc G, Antoine EC, Cajfinger F, Brunet-Pommeyrol A, Agazia C, Khayat D. Management of advanced prostate cancer. Oncologist 2000;5:36-44.

Herbst WP. Effects of estradioi diproprionate and diethylstilbestrol on malignant prostatic tissue. Trans Am Assoc Genitourin Surg 1941;34:195-99.

Cox RL, Crawford ED. Estrogens in the treatment of prostate cancer. J Urol l995;l54:l99l-8.

Smith DC, Redman BG, Flaherty LE, Li L, Strawderman M, Pienta KJ. A phase II trial of oral diethylstilbesterol as a second-line hormonal agent in advanced prostate cancer. Urology 1998;52:257-60.

Leuprolide Study Group. Leuprolide versus diethylstilbestrol for metastatic prostate cancer. N Engl J Med 1984;311:1281-6.

Kiloze SS, Miller MF, Spiro TP. A randomized, comparative study of buserelin with DES/orchiectomy in the treatrnent of stage 02 prostatic cancer patients. Am J Clin Oncol l988;2:~li6-82.

Peeling WB. Phase Itl studies to compare goserelin (Zoladex) with orchiectomy and with diethylstilbestrol in treatment of prostate carcinoma. Urology 1989;33:45-52.

Delaere KP, Van Thilio EL. Rutamide monotherapy as primary treatment in advanced prostatic carcinoma. Semin Oncol 1991 ; 1 8: 13-8.

Tyrrel CJ. Casodex: A pure non-steroidal antiandrogen used as monotherapy in advanced prostate cancer. Prostate 1992;4:97-104. Sogani F'C, Vagahvala MR, Whitmore WF. Experience with flutamide in patients with advanced prostatic cancer without prior endocrine therappy. Cancer 1984;54:744-50.

Lund F, Fasrnussen F. Flutamide versus stilbestrol in the management of advanced prostatic cancer, a controlted prospective study. Br J Uroi 1988;61:140-2.

Staiman VR, Lowe FC. Tamoxifen for flutamidelfinasteride-induced gynecomastia. Urology 1997;50:929-33.

Brufsky A, Fontaine-Rdhe P, Berlane K, Rieker P, Jiroutek M, Kaplan 1, Kaufman D, Kantoff P. Finasteride and flutamide as potency-sparing androgen-ablative therapy for advanced adenocarcinorna of the prostate. Urotogy 1997;49:913-20.

Ornstein DK, Rao GS, Johnson 6, Charlton ET, Andriole GL. Combined finasteride and flutamide therapy in men with advanced prostate cancer. Urology l996;48:9O 1-5.

Mantz CA, Nautiyal J, Awan A, Kopnick Ml Ray P, Kandel G, Niederberger. Potency presewation following conformal radiotherapy for Iocalized prostate cancer: impact of neoadjuvant androgen blockade, treatment technique, and patient-reiated factors. Cancer J Sci Am 1999;5:230-6.

Schroder FH, Collette L, de Reijke TM, Whelan P. Prostate cancer treated by anti-androgens: is semial function preserved? EORTC Genitourinary Group. European Organization for Research and Treatment of Cancer. Br J Cancer 2000;82:283-90.

Friedman G, Lamoureux E, Sherker AH. Fatal fulminant hepatic failure due to cyproterone acetate. Dig Dis Sci 1999;44:1362-3.

Cetin M, Demirci O, Unal A, Altinbas Ml Guven M, Unluhizarci K. Frequency of flutamide induced hepatotoxicity in patients with prostate carcinoma. Hum Exp Toxicol 19W;l8: 137-40.

Wilt TJ, lshani A, Rutks 1, MacDonald R. Phytotherapy for benign prostatic hyperplasia. Public Health Nutr 2000;3:459-72.

Kao GD, Devine P. Use of complementary health practices by prostate carcinoma patients undergoing radiation therapy. Cancer 2000;88:615-9.

Lippert MC, McClain R, Boyd JC,Theodorescu O. Alternative medicine use in patients with localized prostate carcinoma treated with curative intent. Cancer 1999 ;86:2û@-8. Nagata C, Kabuto M, Kurisu Y, Shimizu H. Decreased serum estradiol concentration associated with high dietary intake of soy products in premenopausal Japanese women. Nutr Cancer 1997;29:228-33.

Witte JS, Ursin G, Siemiatycki J, Thompson WD, Paganini-Hill A, Haile RW. Diet and premenopausal bilateral breast cancer: a case-control study. Breast Cancer Res Treat 1997;42:243-51.

Barnes S, Peterson TG, Coward L. Rationate for the use of genistein- containing soy matrices in chemoprevention trials for breast and prostate cancer. J Cell Biochem Suppl l995;22:181-7.

Ng TB, Liu F, Wang ZT. Antioxidative activity of natural products from plants. Life Sci 2000;66:709-23.

Sahin MB, Perman SM, Jenkins G, Clark SS. Perillyl alcohol selectively induces GOIG1 arrest and apoptosis in BcrtAbl-transformedmyeloid cel! lines, Leukemia 1999;13:1581-91.

Satomi Y, Miyamoto S, Gould MN. Induction of AP-1 activity by perillyl alcohol in breast cancer cells. Carcinogenesis t999;20:1957-61.

Drees M, Dengler WA, Roth T, Labonte H, Mayo J, Maispeis t, Grever M, Sausville EA, Fiebig HH. Flavopiridol (L86-8275): seiective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clin Cancer Res 1997;3:273-9.

Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. FIavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res 1996;56:2973-8.

Li Y, Bhuiyan M, Aihasan S, Senderowicz AM, Sarkar FH. Induction of apoptosis and inhibition of c-erbB-2 in breast cancer cells by flavopiridol. Clin Cancer Res 2000;6:223-9.

Ripple GH, Wilding G. Drug development in prostate cancer. Semin Oncol 1999;26:217-26.

Shapiro GI, Koestner DA, Matranga CB, Rollins BJ. Flavopiridol induces celI cycle arrest and p53-independent apoptosis in non-small ceil lung cancer ceIl lines. Clin Cancer Res 1999;5:2925-38.

Sun X-Y, Plouzek CA, Henry JP, Wang lTY, Phang JM. Increased UDP- giucuronosyl-transferase activity and decreased prostate specific antigen production by biochanin A in prostate cancer cePs. Cancer Res 1998;58:2379-84.

Lu R, Serrero G. Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells. J Cell Physiol 1999; 1?9:29?-304.

Heisler LE, Evangelou A, Lew AM, Trachtenberg JI Elsholtr HP, Brown TJ. Androgendependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor. Molec Cell Endocrinol 1997; 126:59-73.

Reyes-Moreno C, Frenette G, Boulanger J, Lavergne E, Govindan MV, Koutsilieris M. Mediation of glucocorticoid receptor function by transforming growth factor beta I expression in human PC-3 prostate cancer cells. Prostate 1995;26:260-9.

Lau KM, LaSpina Ml Long JI Ho SM. Expression of estrogen receptor (ER)- alpha and ER-beta in normal and malignant prostatic epithelial cells: reguiation by rnethylation and involvement in growth regulation. Cancer Res 2000;60:3175-82.

Hobisch A, Hittmair A, Daxenbichler O, Wille SI Radmayr Cl Hobisch-Hagen P, Bartsch G,Klocker H, Culig 2. Metastatic lesions from prostate cancer do not express oestrogen and progesterone receptors. J Pathol 1997; 182956- 61.

Magklara A, Grass L, Diamandis EP. Differential steroid hormone regulation of human glandular kallikrein (hW) and prostate-specific antigen (PSA) in breast cancer cell lines. Breast Cancer Res Treat 2000;59:263-70.

Rao PN, Wang 2, Cessac JW, Rosenberg RS, Jenkins DJ, Diamandis EP. New 11 beta-aryl-substituted steroids exhi bit bath progestational and antiprogestational activity. Steroids 1998;63:523-30.

Eisenberg DM, Davis RB, Ettner SL, Appel S, Wilkey SIVan Rompay M, Kessler RC. Trends in alternative medicine use in the United States. JAMA 1998;28O:l569-75.

Eisenberg DM, Kessler RC, Foster C, Nodock FE, Calkins DR, Delbanco TL. Unconventional medicine in the United States. Prevalence, costs, and patterns of use. N Engl J Med 1993;328:246-52.

Radimer KL, Subar AF, Thompson FE. Nonvitamin, nonmineral dietary supplements: issues and findings from NHANES III. J Am Diet Assoc 2000; 100:447-54. Spaulding-Albright N. A review of some herbal and related products commonly used in cancer patients. J Am Diet Assoc 1997;97:S208-15.

Carbin BE, Larsson B, Lindhal O. Treatment of benign prostatic hyperplasia with phytosterols. Br J Urol 1990;66:63941.

Champault G, Patel JC, Bonnard AM. A double-blind trial of an extract of the plant Serenoa repens in benign prostatic hyperplasia. Br J Clin Pharmacol 19841 8:461-2.

Wilt TW,lshani A, Stark G, MacDonald RI Lau JI Mulrow C. Saw palrnetto extracts for treatrnent of benign prostatic hyperplasia. JAMA l998;28O: 1604- 9.

Shaw CR. The perimenopausal hot flash: epidemiology, physiology, and treatment. Nurse Pract 1997;22:55-6, 61 -6.

Zhu DP. Dong quai. Am J Chin Med 1987;15:117-25.

Mindell E. Anti Aging Bible 1996.

Wright JV. Hormones for menopause. Nutr Healing 1996;June:l-2,9.

Baum JA, Teng Hl Erdman JW Jr, Weigel RM, Klein BPI Persky WV, Freels SI Surya Pl Bakhit RM, Ramos El Shay NF, Potter SM. Long-term intake of soy protein improves blood lipid profiles and increases mononuclear cell low- density-lipoprotein receptor messenger RNA in hypercholesterolemic, postmenopausal women. Am J Clin Nutr l998;68:545-51.

Potter SM, Baum JA, Teng Hl Stillman RJ, Shay NF, Erdman JW Jr. Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr 1998;68:1375S-13798.

Chiechi LM. Dietary phytoestrogens in the prevention of long-term postmenopausal diseases. Int J Gynaecol Obstet 1999;67:39-40.

The role of isoflavones in menopausal health: consensus opinion of The North American Menopause Society. Menopause 2000;7:215-29.

Scambia G, Mango O, Signonle PG, Angeli RA, Palena C, Gallo O, BombardeIli E, Morazzoni Pl Riva A, Mancuso S. Clinical effects of a standardized soy extract in postmenopausal women: a pilot study. Menopause 2000;7:10S-11.

Sun LZ, Currier NL, Miller SC. The American coneflower: a prophylactic rote involving nonspecific immunity. J Akern Complement Med 1999;5:43746. Borchers AT, Keen CL, Stern JS, Gershwin ME. Inflammation and Native American medicine: the role of botanicals. Am J Clin Nutr 2000;72:339-347.

Stevinson Cl Ernst E. A pilot study of Hypericum perforatum for the treatment of premenstrual syndrome. BJOG 2000;107:870-6.

Brenner RI Azbel V, Madhusoodanan S, Pawlowska M. Cornparison of an extract of hypedcum (LI 160) and sertraline in the treatment of depression: a double-blind, randomized pilot study. Clin Ther 2000;22:411-9.

Henig YS, Leahy MM. Cranberry juice and urinary-tract health: science supports folklore. Nutrition 2000;16:684-7.

Wilson T, Porcari JP, Harbin D. Cranberry extract inhibits low density lipoprotein oxidation. Life Sci 1998;62:381-6.

Nuttall SL, Kendall MJ, Bombardelli E, Morazzoni P. An evaluation of the antioxidant activity of a standardized grape seed extract, Leucoselect. J Clin Pharm Ther 1998;23:385-9.

Bagchi Dl Garg A, Krohn RL, Bagchi Ml Tran MX, Stohs SJ. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res Commun Mol Pathol Pharmacol 1997;9S:l79-89.

Zhao J, Wang JI Chen Y, Agarwal R. Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the rnouse skin two-stage initiation-promotion protocol and identification of procyanidin 05-3'-gallate as the most effective antioxidant constituent. Carcinogenesis 1999;20:1737-45.

Moyad MA. Alternative therapies for advanced prostate cancer. What should I tell my patients? Urol Clin North Am 1999;26:413-7.

Bomser J, Madhavi DL, Singletary K, Smith MA. In vitro anticancer activity of fruit extracts from Vaccinium species. Planta Med 1996;62:212-6.

Rosenberg Zand RS, Grass L, Magklara A, Jenkins DJA, Diamandis €P. Is ICI 182,780 an anti-progestin in addition to being an anti-estrogen? Breast Cancer Res Treat 2OOO;60:1-8.

Hunter MS, O'Dea 1. Perception of future health risks in mid-aged women: estimates with and without behavioural changes and hormone replacement therapy. Maturitas 1999;33:37-43.

Tan RS, Philip PS. Perceptions of and risk factors for andropause. Arch Androl 1999;43:227-33. Moyad MA, Pienta KJ, Montie JE. ilse of PC-SPES, a commercially available supplement for prostate cancer, in a patient with hormone-naive disease. Urology 1999;54:319-23.

PromensilTMinformation insert, Novogen Ltd.

The effects of isoflavone phytoestrogens on bone; preliminary results from a large randomised controlled trial. Atkinson C, Compston JE, Robins SPI Bingham SA. Endo 2000, The Endocrine Society 82" Annual Meeting, Toronto, ON, June 21 -24,2000.

Paganga G, Miller N, Rice-Evans CA. The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute? Free Radic Res 1999;30:153-62.

Agarwal C, Sharma Y, Zhao J, Agarwal R. A poiyphenoiic fraction from grape seeds causes irreversible growth inhibition of breast carcinoma MDA-MB468 cells by inhibiting mitogen-activated protein kinases activation and inducing G1 arrest and differentiation. Clin Cancer Res 2000;6:2921-30.

Gali HU, Perchellet EM, Gao XM, Karchesy JJ, Perchellet JP. Cornparison of the inhibitory effects of monomeric, dimeric, and trimeric procyanidins on the biochemical markers of skin tumor promotion in mouse epidermis in vivo. Planta Med 1994;60:235-9.

Charnomile gelcaps information insert, Jamieson Natural Sources.

Zava DT, Dollbaum CM, Blen M. Estrogen and progestin bioactivity of foods, herbs, and spices. Proc Soc Exp Biol Med l998;2li:369-78.

Hirata JD, Swiersz LM, Zell B, Small Fi, Ettinger B. Does dong quai have estrogenic effects in postmenopausal women? A double-blind, placebo- controlled trial. Fertil Steril 1997;68:98 1-6.

Goepel Ml Hecker U, Krege S, Rubben Hl Michel MC. Saw palrnetto extracts potently and noncompetitively inhibit human alphal-adrenoceptors in vitro. Prostate l999;38:208-15.

Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, Heubi JE. Bioavaitability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr 2001;131:1362S-75s.

Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, Ogawa S, [noue S, Muramatsu M, Masamune Y. Interaction of phytoestrogens with estrogen receptors alpha and beta. Biol Pham Bull 2001 ;24:351-6. Franke AA, Custer W, Cema CM, et al. Rapid HPLC analysis of dietary photoestrogens from leg urnes and from human urine. Proc Soc Exp Med 1995;208:18-26.

Setchell KD, Borriello SP, Hulme P,Kirk DN, Axelson M. Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease. Am J Clin Nutr 1984;40:569-78.

Adlercreutz H. Phytoestrogens: Epidemiology and a possible role in cancer protection. Environ Healt h Perspect 1995;l O3:lO3-l2.

Barnes S. Effect of genistein in in vitro and in vivo models of cancer. J Nutr 1995;125 :7773-83.

Messina M, Barnes S, Setchell KD. Phytosestrogens and breast cancer. Lancet lWi;3SO:971-2.

Anderson JW, Johnstone BM, Cook-NeweIl ME. Meta-analysis of the effects of soy protein intake on serum iipids. N Engl J Med 1995;333:276-82.

Anthony MS, Clarkson TB, Bulfock BC, Wagner JO. Soy protein versus soy phytoestrogens in the prevention of diet-induced coronary artery atherosclerosis of male cynomolgus monkeys. Arterioscler Thmb Vasc Biol 1997; 1?:2524-31.

Wei Hl Wei L, Frenkel K, Bowen R, Bames S. Inhibition of tumor promoter- induced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr Cancer 1993;2O:l-l2.

Setchell KD. Phytoestrogens: The biochemistry, physiology, and implications for human health of soy isoflavones. Am J Clin Nutr 1998;68:1333S-46s.

Adlercreutz H. Diet, breast cancer, and sex hormone metabolism. Ann NY Acad Sci 1WO;595:28l-W.

Willard ST, Frawley LS. Phytoestrogens have agonistic and combinational effect on estrogen-responsive gene expression in MCF-7 human breast cancer cells. Endocrine 1998;8:? 17-21.

Davis DL, Axelrod O, Bailey L, Gaynor M, Sasco AJ. Rethinking breast cancer risk and the environment: the case for the precautionary principle. Environ Health Perspect 1998; 1Q6:523-9.

Welshons WV, Murphy CS, Koch R, Calaf G, Jordan VC. Stimulation of breast cancer cells in vitro by the environmental estrogen enterolactone and the phytoestrogen equol. Breast Cancer Res Treat 1987;10:169-75. Hilakivi-Clarke L, Cho El Clarke R. Maternal genistein exposure mimics the effects of estrogen on mammary gland development in female mouse offspring. Oncol Rep l998;5:609-16.

Divi RL, Chang HC, Doerge DR. Anti-thyroid isoflavones from soybean: Isolation, characterization, and mechanisms of action. Biochem Pharmacol 1997;54:1087-96.

Fort Pl Moses N, Fasano M, Goldberg T, Lifshitz F. Breast and soy-formula feedings in early infancy and the prevalence of autoimmune thyroid disease in children. J Am Coli Nutr 1990;9:164-7.

Freni-Titulaer LW, Cordero JF, Haddock L, Lebron G, Martinez R, Mills JL. Premature thelarche in Puerto Rico. A search for environmental factors. Am J Dis Child 1986;14O:l263-7.

Anonymous. Soy infant formula should be restricted, says New Zealand Ministry. Washington DC, CRC Press, Food Labeling Nutrition News 1998; NOV.1 1 :9-10.

Food and Drug Administration Final Rule for Food Labeling: Health Clinicals: Soy Protein and Coronary Heart Disease. No. 279, November 10,1999.

Jenkins DJ, Kendall CW, Mehling CC, Parker Tl Rao AV, Agarwal S, Novokmet R, Jones PJ, Raeini Ml Story JA, Furumoto E, Vidgen El Griffin LC, Cunnane SC, Ryan MA, ConnelIy PW. Combined effect of vegetable protein (soy) and soluble fiber added to a standard cholesterol-lowering diet. Metabolism 1999;48:809-16.

Diamandis EP, Yu Hl Melego DN. Ultrasensitive prostate-specific antigen assays and their clinical application. Clin Chem 1996;42:853-7.

Wang H-J, Murphy P. lsoftavone composition of American and Japanese soybeans in Iowa: Effects of variety, crop year, and location. J Agric Food Chem 1994;42:1674-7.

SAS Institute. SAS.STAT User's Guide (ed 6-12) Cary, NC, SAS Institute, 1997.

Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner 8, Willett WC. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993;328:14M-9.

Rimm EB, Starnpfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med l993;328: 1450-6. Esterbauer H, Striegl G, Puhl H, Oberreither S, Rotheneder M, el-Saadani Ml Jurgens G. The role of vitamin E and carotenoids in preventing oxidation of low density lipoproteins. Ann N Y Acad Sci 1989;570:254-67.

Rao AV, Agarwal S. Bioavailability and in vivo antioxidant properties of lycapene from tomato products and their possible role in the prevention of cancer. Nutr Cancer 1998;31:199-2O3.

Kritchevsky D. Dietary protein, cholesterol and atherosclerosis: A review of the early history. J Nutr 1995;125:589S-93S.

Carroll KK. Review of clinical studies on cholesterol-lowering response to soy protein. J Am Diet Assoc 199 1;91:820-7. tiodgson JM, Puddey IB, Beilin LJ, Mori TA, Croft KD. Supplementation with isoflavonoid phytoestrogens does not alter serum lipid concentrations: a randornized controlled trial in humans. J Nutr I998;l28:728-32.

Bennetts HW, Undennrood EJ, Shier FL. A specific breeding problem in sheep in subterranean clover patures in Western Australia. Aust J Agric Res 1946;22:131-8.

Slavin JL, Karr SC, Hutchins AM, Lampe JW. Influence of soybean processing, habitua1 diet, and soy dose on urinary isoflavonoid excretion. Am J Clin Nutr l998;68:lM!S-I495S.

Cummings SR, Eckert S, Krueger KA, Grady D, Powles TJ, Cauley JA, Norton L, Nickelsen T, Bjamason NH, Morrow M, Lippman ME, Black D, Glusman JE, Costa A, Jordan VC. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 1999;281:2189-97.

Fisher BI Dignam J, Wolmark Nt Wickerham DL, Fisher ER, Mamounas E, Smith R, Begovic M, Dimitrov NV, Margolese RG, Kardinal CG, Kavanah MT, Fehrenbacher L, Oishi RH. famoxifen in treatment of intraductal breast cancec National Surgical Adjuvant Breast and Bowel Project 6-24 randomised controlled trial. Lancet 1999;353;1993-2000.

Jialal Il Grundy SM. Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein. J Lipid Res 1992;33:899-906.

Adlercreutz H. Epidemiology of phytoestrogens. Baillieres Clin Endocrinol Metab 1998; 12:605-23. 453. Stephens FO. The rising incidence of breast cancer in women and prostate cancer in men. Dietary influences: a possible preventive role for nature's sex hormone modifiers - the phytoestrogens. Oncol Rep 1999;6:865-70. 454. Persky V, Van Horn L. Epidemiology of soy and cancer: perspectives and directions. 3 Nutr 19953125:709S-7128.

455. Moyad MA. Soy, disease prevention, and prostate cancer. Semin Urol Oncol 1999; l7:97-lO2.

456. Pollard Ml Wolter W. Prevention of spontaneous prostate-related cancer in lobund-wistar rats by a soy protein isolate/isoflavone diet. Prostate 2000;45:101-5.

457. Xu X, Duncan AM, Wangen KE, Kurzer MS. Soy consumption alters endogenous estrogen metabolism in postmenopausal wornen. Cancer Epidemiol Biomarkers Prev 2000;9:781-6.

APPENOIX

Chemical Structures of Flavonoids and Related Compounds

Structure 7,8-Benzoflavone

Biochanin A

Caffeic acid

Caffeine Daidzein HO

2',4'-Dihydroxyacetophenone

""OH CHO 3,4-Dimethoxycinnamic acid

7,8-Dimethoxyflavone

Ellagic acid Flavone

O

Folic acid O OH

OHN ,, O II \ - II HO-c-&H~ CH2-C-OH NH2 Gdlic acid

Gardenin

Genistein

HarmaIine 2'-Hydroxyflavanone langostine Morin Picrotin