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ANSTATE UIN VERSI TYL I III |IIIII|1II|1I1II 193 01712 9093 2; II1|I1IIII1II|II This is to certify that the dissertation entitled Genistein and Mammary Tumorigenesis presented by Ross C. Santell has been accepted towards fulfillment of the requirements for Ph.D. degree in Nutritional Biochemistry ,444/ //€ ,J v Major p fessor Date 26 Nov. 1997 MS U is an Afflrmatiw Action/Equal Opportunity Institution 0-12771 I LIBRARY Michigan State i University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE 1M CUMJGS-p.“ Genistein and Mammary Tumorigenesis BY Ross C. Santell .A DISSEREAIION Submitted to Michigan State University in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1997 ABSTRACT Genistein and Mammary Tumorigenesis BY Ross C. Santell Soybeans contain a high level of isoflavonesincluding genistein, daidzein and glycetein. I have focused on genistein due to its estrogenic and antiproliferative activities which could affect mammary tumorigenesis. Dietary genistein (750 ug/g American Institute of Nutrition semi-purified diet, AIN-76A) increased uterine weight and uterine c—fos mRNA expression, increased plasma prolactin, and stimulated lobular/alveolar development of the mammary gland in ovariectomized Sprague Dawley rats. The plasma concentration of total (free + conjugated) genistein was 2.54 uM. Competitive binding studies indicated that genistein competes with estradiol for the estrogen receptor with an affinity approximately l/lOOth that of estradiol. However, genistein (750 ug/g AIN-76A) did not antagonize the action of concurrently fed estradiol (1.0 ug/g AIN-76A) in ovariectomized rats or in intact peripubertal rats fed through 30-44 days of age. Genistein exerts antiproliferative effects on both estrogen dependent and independent cell lines. I elected to study the effects of genistein on the growth of estrogen independent MDA-MB-23l human breast cancer cells. Genistein (20 uM) inhibited cell proliferation in_yitrg by 50%. The cell cycle was blocked at Cfi/M when 40 uM or 80 uM genistein was added to the medium. To evaluate the effect of genistein in_yiyg, female athymic mice were inoculated with cells and fed genistein (750 ug/g AIN-93G). Genistein at this dose did not affect tumor growth. Genistein (3000 ug/g AIN-93G) was then fed to tumor bearing mice. This dose inhibited tumor growth when compared to untreated control mice; however, there was approximately a 10% reduction in food intake in the genistein group. Weight gain did not differ. Total plasma genistein concentration was 5.88 uM. The effect of dietary genistein on initial tumor development was studied by feeding mice 750 ug genistein/g AIN-93G before tumor cell inoculation. This dose of genistein did not inhibit tumor development or growth. This research demonstrates that dietary genistein is estrogenic in estrogen responsive tissues in ovariectomized rats. Genistein inhibited cell growth in culture by blocking the cell cycle at Gwa In.addition dietary genistein (3000 ug/g AIN-9BG) inhibited MDA-MB-231 cell growth in athymic mice. .Acknowledgmnnts I would like to thank all the friends I have made during the pursuit of this degree. Your friendships have been invaluable through the good times and the bad. I would also like to thank my committee members for their help, especially Bill for his support and encouragement. Special thanks to you Mom for your encouragement and love. I would not be here today without your love and support. Also to my sisters, especially you Susan, thank you for your love and encouragement. Most of all thanks to you Candy for your love, support and patience over the many years. iv Table of Contents INTRODUCTION CHAPTER I. REVIEW OF THE LITERATURE A. B. 1. 2. [—1 wa WNH NH Cancer Epidemiology Breast cancer incidence Diet and cancer a. Soybeans and cancer 1. Protease inhibitors 2. Phytic acid 3. Isoflavones Genistein Estrogenic activity of genistein a. Uterotrophic effects of genistein b. Mammatrophic effects of genistein c. Antiestrogenic effects of genistein Antioxidant activity of genistein Genistein inhibits topoisomerase II Genistein inhibits tyrosine kinases Breast Cancer Etiology Genistein and initiation Genistein and promotion Genistein and progression Antiproliferative Effects of Genistein Human Consumption of Genistein Justification of the Models Rat model Athymic mouse model Summary CHAPTER II. DIETARY GENISTEIN EXERTS ESTROGENIC ....... 47 EFFECTS UPON THE UTERUS, MAMMARY GLAND AND THE HYPOTHALAMIC/PITUITARY AXIS (Published in the Journal of Nutrition 1997. 127(2): 263-269 A. Abstract ....... 47 B. Introduction ....... 48 C. Materials and Methods ....... 50 1. Chemicals ....... 50 2. Animals ....... 51 3. Diets ....... 51 4. Experimental design ....... 52 5. Analysis of uteri ....... 53 6. Analysis of mammary glands ....... 54 7. Northern blot analysis ....... 55 8. Plasma prolactin analysis ....... 56 9. Competitive binding analysis ....... 57 10. Serum genistein analysis ....... 58 11. Statistical analysis ....... 59 D. Results ....... 60 1. Competitive binding analysis ....... 60 2. Uterotrophic effects of dietary ....... 6O genistein 3. Induction of c-fos ....... 62 4 Mammatrophic effects of dietary ....... 7O genistein 5. Plasma prolactin analysis ....... 71 6. Serum genistein analysis ....... 71 E. Discussion ....... 72 1. Competitive binding analysis and ....... 72 induction of c-fos 2. Uterotrophic effect of dietary ....... 73 genistein 3. Effects of dietary genistein on ....... 74 the mammary gland and plasma prolactin 4. Serum genistein analysis ....... 77 F. Acknowledgments ....... 79 vi CHAPTER III. GENISTEIN INHIBITS ESTROGEN RECEPTOR NEGATIVE HUMAN BREAST CANCER CELL GROWTH lN_¥lIBQ AND IN_MIMQ ....... 80 A. Abstract ....... 80 B. Introduction ....... 82 C. Materials and Methods ....... 85 1. Chemicals ....... 85 2. Animals and diets ....... 85 3. Experimental design ....... 86 a. Cell proliferation ....... 86 b. Flow cytometry ....... 87 c. Mice fed 0-6000 ug genistein/g diet ....... 89 d. Genistein fed to mice inoculated ....... 90 with MDA-MB-231 cells e. High pressure liquid chromatography ....... 92 analysis of plasma genistein f. Statistical analyzes ....... 93 D. Results ....... 95 1. In 11:19 MDA-MB-231 cell proliferation ....... 95 and cell cycle analysis 2. Dose response study of dietary ....... 95 genistein on plasma genistein and food intake 3. Effect of Genistein Fed to Mice ....... 99 Inoculated with MDA-MB-231 Cells E. Discussion ...... 106 SUMMARY AND FUTURE RESEARCH ...... 112 Summary ...... 112 Future Research ...... 117 APPENDICES ...... 120 Introduction to the Appendices ...... 121 Appendix A ...... 122 Effect of Genistein, with 1% or 10% Fetal Bovine Serum, on MDA-MB—231 Cell Proliferation vfi Appendix B Kinetic Analysis of B—Glucoronidase ...... 127 Appendix C Food Consumption Pattern in the Mouse ...... 130 Appendix D Athymic Mice Fed Genistein for 10 Weeks ...... 133 Appendix E Plasma Analysis of Genistein from Mice ...... 137 Given a Bolus of 6 mg Genistein Appendix F Gavage Vehicle and Its Affect on Food ...... 140 Intake in the Mouse Appendix G Genistein Gavaged to Tumor Bearing Mice ...... 143 Appendix H Genistein Release from Elvax Pellets ...... 148 Appendix I Effect of Genistein Pellet Implants on ...... 150 Mammary Gland Development in the Mouse Appendix J Genistein/Cholesterol Pellet Implant ...... 155 into Tumor Bearing Mice Appendix K Genistein Pellet Implanted into Mice ...... 158 List of References ...... 160 i/iii List of Tables TABLE 1. Effect of dietary genistein and estradiol on uterine weight in ovariectomized mature ratS° Table 2. Effect of dietary genistein and estradiol on uterine weight and plasma prolactin in adult ovariectomized rats Table 3. Effect weight of intact Table 4. Effect estradiol on the adult rats of dietary genistein on uterine immature rats of dietary genistein and mammary gland of ovariectomized ....... 64 ....... 65 ....... 66 ....... 67 Table 5. Effect of Genistein Upon the Cell Cycle ....... 96 of MDA-MB-231 Cells. ix List of Figures Figure 1: Competitive binding analysis of ....... 63 genistein and estradiol using rat uterine cytosol Figure 2: Effect of dietary genistein and ....... 68 estradiol upon the uterine expression of c—fos in ovariectomized mature rats Figure 3: Photographs of the mammary glands from ....... 69 rats treated with dietary genistein and estradiol Figure 4. Effect of Genistein on MDA—MB-231 Cell ....... 94 Proliferation Figure 5. Effect of Genistein Upon the Cell ....... 98 Cycle of MDA-MB-231 Cells Figure 6. Plasma Genistein Concentration in Mice ....... 100 Fed Genistein Figure 7. Tumor Growth in Mice Fed 750 ug/g Genistein After Tumor Formation Figure 8. Tumor Growth in Mice Fed 3000 ug/g Genistein After Tumor Formation Figure 9. Tumor Growth in Mice Fed 750 ug/g Genistein Before Tumor Formation Appendices Figures Figure A1. Effect of genistein on MDA-MB-231 Cell Proliferation in 10% fetal bovine serum. Figure A2. Effect of genistein on MDA-MB-231 Cell Proliferation in 1% fetal bovine serum. Figure B. Kinetic analysis of B-glucuronidase in serum from rats gavaged with genistein Figure C. Food intake of mice over a 24 hour period Figure D. Food intake and weight of mice fed 0, 1500 and 3000 pg genistein/g food xi ...... 102 ...... 104 ...... 105 ...... 125 ...... 126 ...... 129 ...... 131 ...... 135 Figure B. Plasma genistein in mice gavaged ...... 139 with 6 mg genistein Figure F. Food intake in mice gavaged with corn ...... 142 oil or DMSO. Figure 61. Tumor area change ...... 144 Figure 62. Plasma genistein in mice ...... 145 Figure G3. Food intake ...... 146 Figure B. Genistein release from an Elvax pellet ...... 149 Figure 11. Effect of genistein pellet implant on ...... 152 the mammary gland in ovariectomized mice Figure 12. Effect of genistein pellet implant on ...... 153 the mammary gland in intact mice xfi INTRODUCTION This dissertation presents data on the soy phytoestrogen genistein and its effect on estrogen responsive tissues in the female Sprague Dawley rat. Genistein’s effect on estrogen independent MDA-MB-231 human breast cancer cell growth was studied in cell culture and in athymic female mice inoculated with MDA-MB-231 cells. The impetus for these studies came from epidemiological data that point out a geographic variation in the site specific incidence of various cancers (Kodama et al. 1991, Staszewski et al. 1971, Wynder & Hirayama 1977). For example, the incidence of mammary cancer is much higher in US women than it is in Japanese women. It is also clear from the epidemiological data that, in immigrant populations, the site specific tumor incidence present in a home country will, over time, change to that of the host country (Staszewski et al. 1971). For example investigators have shown, that in each of two subsequent generations of Japanese immigrants to the US the incidence of mammary cancer in women has risen to levels approaching that in the US (Buell 1973). These data suggest that environmental factors, including diet, may be largely responsible for the change in tumor incidence patterns. Dietary habits of 2 immigrant populations also tend to change over time, eventually reflecting those in the host country. For example the dietary habits of Japanese migrants to Hawaii change from one in which vegetables (including soybeans)‘are primarily consumed with little meat, as in their home country, to one of less vegetable consumption, including soy, and more meat common in the US (Hankin et al. 1975). Thus the change in dietary habits that occurs concurrently with changes in site specific tumor incidence suggest that diet could play a major role in the etiology of this disease. A review of the literature covering the epidemiology and etiology of cancer and the role of the diet, especially soybeans and specifically the soy phytoestrogen genistein, in these processes is presented in Chapter 1 of the dissertation. Genistein has estrogenic activity in uterine tissue and has been shown to antagonize estrogen in this tissue (Folman & Pope 1966); however, its affect upon the mammary gland and the hypothalamic/pituitary axis, with respect to plasma prolactin concentration, in adult rats is unknown. It is clear that estradiol is required for mammary gland development and is also required for estrogen dependent mammarytumorigenesis in many species. Therefore, compounds with estrogenic activity such as genistein may promote 3 mammary gland development and may also stimulate estrogen dependent mammary tumorigenesis. Our overall objective in the studies presented in Chapter 2 was to determine the in 1129 activity of dietary genistein by analyzing its effect on estrogen responsive tissues in the rodent. It is our hypothesis that dietary genistein will exert estrogenic effects in the uterus, mammary gland and the hypothalamic/pituitary axis. Results from experiments conducted to ascertain the estrogenic activity of various doses of dietary genistein in estrogen responsive tissues in ovariectomized and intact female rats are presented in Chapter 2. During the time the studies in chapter 2 were being conducted there were additional reports on genistein’s ability to inhibit the in_yit;g proliferation of both estrogen dependent and independent cells. I decided to pursue the antiproliferative effects of genistein. Since the antiproliferative effects were observed in estrogen dependent and independent cells I decided to conduct our in 21119 and in yivg studies using estrogen independent MDA-MB- 231 human breast cancer cells. By using estrogen receptor negative cells the potential estrogenic activity of genistein in affecting estrogen dependent tumor formation and growth is eliminated. Cell culture studies were 4 conducted to determine a possible mechanism for genistein inhibiting tumor cell growth and animal studies were conducted to determine whether similar effects could be observed in animals. Our overall objective in these studies was to determine the effect of genistein on the growth of MDA-MB—231 cells in_xi;rg and in_xiyo. It is our hypothesis genistein will inhibit the in_yitrg proliferation of MDA-MB— 231 cells and the in_xivg growth of MDA-MB-231 cell inoculated into the athymic mouse. Results from experiments conducted on genistein’s effect on the proliferation and cell cycle of MDA-MB-231 cells in_yitzg and the growth of these cells in athymic mice is presented in Chapter 3. A number of preliminary studies not included in chapters in the dissertation were conducted to assist in methods development and designing future experiments. Procedures and data from these studies are presented in the appendices. Chapter 1 Literature Review .A. Cancer Epidemiology Data from population studies show a wide geographical variation in the incidence of site specific tumors, suggesting that environmental factors play a major role in the etiology of this disease. Indeed there are certain cancers that are classified as ‘Western cancers’ due to their increased incidence in the Western world. For example, Kodama et al. (1991), found that bladder cancer is higher in western countries than in Japan. Furthermore, bladder cancer was positively related to the occurrence of other western cancers, notably colon and lung, and negatively related to the occurrence of non-western cancers such as stomach and esophageal. Studies of Japanese immigrants to the US have shown an increase in the incidence of bladder cancer and a decrease in the incidence of stomach cancer with both figures approaching the tumor incidence pattern found in US whites (Kodama et al. 1991). The incidence of stomach cancer is high in Poland whereas the incidence of intestinal and breast cancer is low. However the tumor incidence profile of Polish immigrants to Australia, where tumor incidence patterns are the inverse of 6 that in Poland, eventually changes to those of the host country, again implicating environmental causes (Staszewski et al. 1971, McMichael et al. 1980). Other studies have shown similar trends in immigrants to the US regarding cancer of the prostate, colon, breast, bladder and uterus (Wynder & Hirayama 1977). 1. Breast Cancer Incidence Breast cancer is relatively uncommon in Japan whereas in the US it will afflict one in eight to ten women (Claus et al. 1991, Marshall 1993). Approximately 5—10% of breast cancer is thought to be due to inherited defective genes which predispose women to the disease (Claus et al. 1991). For example mutations in chromosome 17q, the locus of the BRCA1 gene which codes for a protein that functions as a tumor suppressor, are linked to early inherited breast cancer in families (Miki et al. 1994). The remaining 90% of breast cancer incidence is believed to be caused by environmental factors such as diet, lifestyle and exposure to environmental contaminants (Wright 1990, Lynch et al. 1992). As mentioned in the preceding section site specific cancer incidence patterns can change as a population migrates from their home country to the host country: this 7 is also true for breast cancer. Early work (Haenszel & Kurihara 1968), using data from 1959—62 to study cancer incidence patterns in Japanese immigrants to the US, did not find a change in breast cancer yet did find increases in cancer of the colon, pancreas, lung, and ovary. Initially these data lead to speculation that genetic differences in these populations accounted for the difference in breast cancer incidence among these countries; however, later work (Buell 1973) using data from 1969-71 did show an increase in the incidence of breast cancer in female Japanese immigrants, albeit occurring at a slower rate than that for cancers of the colon and lung. There was an increase in breast cancer in first generation offspring (Issei) of Japanese immigrants to the US and a further increase approaching that in the US in the second generation (Nisei)(Buell 1973). The research by Buell, in which breast cancer incidence increased in a population upon migrating, discounted population based genetic differences as the basis for the difference in breast cancer incidence between Japanese and US women. This suggests other factors, such as changes in the environment, including diet, could be responsible. 2. Diet and Cancer Dietary choices made throughout life can affect the well-being of the individual both in terms of their present and future health. Examples of the potential role of diet in carcinogenesis include: ingesting carcinogens or metabolic activation of dietary carcinogenic precursors, dietary manipulation of gut transit time thereby decreasing or increasing exposure, and ingesting compounds that interfere with tumorigenesis thereby inhibiting initiation, promotion or progression. Doll & Peto (1981) suggest that the diet's role in tumorigenesis has been underestimated and that from 10-70% of cancers could be prevented by dietary intervention. Epidemiological studies have shown that the consumption of yellow and green vegetables is inversely related to the incidence of cancer (lklk et al. 1990). Furthermore these same studies have shown a positive correlation between meat and fat consumption and tumor incidence. Epidemiological studies which analyzed a countries breast cancer mortality rate and fat consumption have found that these two factors are positively correlated (Carroll 1975). Studies have sought to determine a mechanism for the relationship between breast cancer and fat by analyzing the relationship between dietary fat and serum estrogen levels, 9 the rationale being increases in serum estrogen can lead to an increase in tumor formation in the estrogen responsive mammary gland. When fat intake in both premenopausal and postmenopausal women was decreased from ca. 40% of dietary calories to ca. 20%, serum estradiol concentration was reduced; however, the reduction occurred concurrently with significant reductions in body weight and caloric intake (Rose et al. 1987, Ingram et al. 1987, Prentice et al. 1990). Androgen aromatization to the estrogens can occur in adipose tissue thus weight loss (decrease in adipose tissue) could account for the decrease in estradiol and the role of dietary fat in this process would be secondary to the decrease in body weight brought about by caloric reduction. So the effect of fat in independently lowering serum estradiol, as opposed to weight loss or caloric reduction, is unclear. Numerous animal studies have shown that as the percent of fat in the diet is increased the incidence of tumor formation also increases. However, by increasing the fat content of the diets in these experiments the diets were no longer isocaloric. This is because the body metabolizes dietary carbohydrate (CHO) and fat differently. For example, in de novo fat synthesis from CHO, CHO must be broken down to acetyl CoA and then the acetyl CoA is used 10 for fat synthesis (an energy requiring process). Dietary fat, on the other hand, is efficiently converted to body fat requiring less energy. So although the diets are isocaloric when determined using Atwater values they are not equivalent metabolically. It is therefore difficult to separate effects due to caloric intake, a reduction of which is known to inhibit tumor development, from fat intake (Welsch 1994). Caloric reduction has been shown to alter the hormonal milieu in rodents and this could affect development of hormone responsive tumors (Boutwell et al. 1948). For example prolactin, estrogen and insulin are decreased in chronically, caloric restricted rodents; whereas, the glucocorticoids are higher (Pariza & Boutwell 1987). Estrogen and prolactin both enhance mammary tumorigenesis and decreasing their concentration, as a result of caloric reduction, could inhibit tumorigenesis. Research in humans exploring the relationship between dietary fat intake and breast cancer has yielded conflicting results. An ongoing prospective study (Willett et al. 1992) involving over 80,000 nurses of which ~1500 had breast cancer, has not found an association between dietary fat intake and the incidence of breast cancer. Fat intake in this study varied from less than 29% to over 49% of calories. A prospective study by van den Brandt et al. 11 (1993) involving over 60,000 postmenopausal women of which 471 cases of breast cancer were available for analysis, has also failed to find an association between total dietary fat and breast cancer. However in this study saturated fat was weakly associated with breast cancer (p = 0.052). In this study fat intake varied from 32% to 46% of total calories. Many other studies have failed to show a link between the amount of fat consumed and breast cancer incidence in humans (Lee et a1. 1991, van den Brandt et al. 1993, Graham 1982, Graham et al. 1991). Contrary to these reports, a prospective study conducted by Howe (1991), involving over 55,000 women of which 519 breast cancer cases were available for analysis, did find a positive association between total fat intake and breast cancer. In this study saturated fat was not associated with breast cancer however monounsaturated fats were associated with breast cancer (p = 0.04). In this study relative risk for breast cancer from dietary fat was actually lower in the second quartile (0.73) and slightly lower in the third quartile (0.98) while in the fourth quartile the relative risk rose to 1.3. There are a number of studies which suggest dietary fat is associated with breast cancer (Marshall et al. 1992, Howe et al. 1991) and a number of studies that have not found an 12 association. At present the role of fat in the development of breast cancer is not clear. It is possible that as migrant populations adopt the dietary habits of their host country, this change in diet eventually gives rise to the site specific tumor incidence pattern seen in the host country. Whether the absence of protective compounds found in their native diet or the presence of harmful compounds in their new diet are responsible for the change in cancer incidence patterns is unknown. It is known however, that as immigrants adopt the new dietary habits of the host country they also give up those of their home country. These changes, with respect to the Japanese, are reflected in the consumption of more meat and fat, and less plant products including soy products (Nomura et a1. 1978, Hankin et al. 1975). Plants contain nutrients and non-nutrients which have demonstrated beneficial effects upon processes thought to be involved in tumorigenesis. For example carotenoids, a group of micronutrients found in vegetables and fruits (Khachik et al. 1995), have demonstrated anticarcinogenic activity in animal experiments (Nishino 1995). Monoterpenes (Gould 1995), found in plants, and polyphenols (Stoner & Mukhtar 1995), found in plants and various teas, have also been shown to inhibit tumorigenesis. Soybeans also contain a 13 number of compounds (discussed below) that have demonstrated anticarcinogenic abilities. Epidemiological studies have found that concurrent changes in tumor incidence and dietary habits accompany a change in geographical location. These observations implicate environmental factors, including diet, in the etiology of breast cancer. This is the premiss for the proposed research: are nutrients consumed in an immigrants home country responsible for the lower incidence of breast cancer and upon migrating, when dietary habits of the home country are no longer followed, the lack of these nutrients responsible for an increase in breast cancer? For example does the consumption of soy products in the traditional Japanese diet offer some protection against breast cancer and when consumption of these products decrease the protective effect is lost? a. Soybeans and Cancer As immigrant populations assimilate into the host country they eventually adopt dietary practices of the host country while at the same time forgo the traditional dietary habits of their home country (Hankin et al. 1975). With regard to the Japanese this is especially true for soy products. Soy products constitute an integral part of the 14 traditional Japanese diet (Wynder & Hirayama 1977, Hankin et al. 1975) with 1-2 servings consumed per day. Research has shown that the consumption of soy inhibits tumorigenesis in rodent models. For example, Barnes et al. (1990) found that powdered soybean chips and autoclaved powdered soybean chips, a process that destroys much of the protease inhibitors (Dipietro & Liener 1989), both led to a decrease in mammary tumor formation in rats subjected to 7,12— dimethylbenz[a]anthracene (DMBA) and nitroso-methyl urea (NMU) chemical carcinogens. Hawrylewicz et al. (1991) showed a similar decrease in tumor incidence when rats were fed a diet containing 19% soy protein isolate. Baggott et al. (1990) also showed similar results in rats treated with DMBA and then fed an AIN-76A diet supplemented with miso (soy soup) at 25%. Troll et al. (1980) has shown that mammary tumor incidence in irradiated rats was reduced by feeding a 50% soybean diet; however, in this study food intake was also decreased. Soybeans and soy products contain compounds such as protease inhibitors, phytic acid and isoflavones which are thought to inhibit tumorigenesis (Barnes et al. 1990, Kennedy & Manzone 1995). The majority of research conducted to date has concentrated on the Bowman-Birk protease inhibitor, and the isoflavone genistein. 15 l. Protease Inhibitors Protease inhibitors are thought to inhibit carcinogenesis by binding to and inhibiting the activity of proteases involved in cell growth (negative transcription factors), secreted by tumor cells, or by decreasing protein absorption by inhibiting intestinal proteases; however, the exact mechanism is unknown (reviewed in Kennedy 1995). Natural protease inhibitors, including the soybean trypsin inhibitor (SBTI), inhibited transformation of irradiated C3H/10Tl/2 mouse embryo fibroblast cells treated with 12-0- tetradecanoylphorbol-13-acetate (TPA) (Kennedy & Little 1981). The authors speculated that the protease inhibitors inhibited the action of TPA induced proteases involved in carcinogenesis. Cell culture experiments have shown that the soybean derived Bowman Birk protease inhibitor (BBI), an 8 kd peptide, will inhibit cytosolic proteases involved in cell growth (Billings & Habres 1992). 881 has also been shown to inhibit transformation of C3H/1OTl/2 cells in vitrQ when administered immediately after carcinogen exposure (St. Clair & St. Clair 1991), or after radiation (Kennedy 1985); however, it is ineffective when given during or days after carcinogen exposure. Furthermore, modified BBI, no longer able to inhibit trypsin activity, was just as effective in inhibiting radiation induced transformation however when 881 16 was modified to no longer inhibit chymotrypsin it lost the ability to inhibit transformation (Yavelow et al. 1985). This suggests that BBI inhibits proteases similar to chymotrypsin. BBI also inhibits the formation of a number of different tumors in laboratory animals treated with a variety of chemical carcinogens (Troll & Kennedy 1989, reviewed in Kennedy 1995). For example, dietary BBI (0.1% of the diet) inhibited dimethyhydrazine (DMH) induced colon cancer in mice whereas autoclaved BBI (a process that destroys BBI) did not suggesting that the BBI was responsible (Billings et al. 1990). Research has shown cytoplasmic localization of dietary derived BBI in intestinal cells leading to speculation that it may be able to inhibit tumorigenesis at sites other than the gastrointestinal tract (Billings et al. 1991, Dipietro & Liener 1989). In fact studies have shown BBI to inhibit tumor formation in the liver and lung suggesting systemic effects (Kennedy 1995). Research by Hirayama (1982) on the relationship between gastric cancer and the consumption of soybean paste soup, has shown significant reductions in gastric cancer in those who consume daily amounts of the soup, a traditional component of the Japanese diet. The authors speculated protease inhibitors could be responsible for the effect. Barnes et al. (1990) found that powdered 17 soybean chips and autoclaved-powdered soybean chips (a process that destroys much of the protease inhibitors) (Dipietro & Liener 1989), both lead to a decrease in mammary tumor formation in rats subjected to DMBA or NMU chemical carcinogens, suggesting compounds other than the protease inhibitors (isoflavones) are responsible for the decrease in tumor incidence. However, Kennedy (1995) points out that the autoclaved soy chips still had a sufficient amount of BBI to inhibit tumorigenesis. The research to date suggests soybean derived protease inhibitors may inhibit tumorigenesis in in vitrg and in yivg models; however their effect upon human carcinogenesis is unknown. 2. Phytic Acid Phytic acid or inositol hexaphosphate (1P6) is a natural antioxidant found in fiber rich foods, including soybeans, and is suspected of contributing to the apparent decrease in colon carcinogenesis in populations consuming high fiber diets (Alabaster et al. 1996). It is present in soybeans at up to 1.4% on a dry weight basis (Graf & Eaton 1990). IP6 is absorbed quickly and distributed to various organs within one hour after ingestion (reviewed in Shamsuddin 1995). Its mode of action is unknown. One hypothesis suggests IP6 inhibits oxidative damage (DNA damage) induced by iron by binding iron and keeping it in 18 the ferric state (Graf & Eaton 1990). Another hypothesis suggests IP6 functions in signal transduction pathways by interacting with the intracellular inositol phosphate-3 (IPQ pool and altering signal transduction (Shamsuddin 1995). IP6 has been shown to inhibit in 11:29 cell growth and azoxymethane induced colon cancer in rats (Shamsuddin 1995). In addition IP6 fed to mice on high-fat-high-calcium or high-fat-high-iron diets reduced the labeling index (indicator of cell proliferation) in the colon and breast to that of the low-fat group (Thompson & Zhang 1991). It was suggested that the calcium and iron were bound by IP6 and that this inhibited tumor growth. This study illustrates an important feature of 1P6, its ability to bind divalent cations. Although there have been very few published studies on the role of soy-derived phytic acid in carcinogenesis, phytates have been studied for their ability to bind divalent cations. Binding of divalent cations, particularly calcium and iron, could inhibit the absorption of these minerals and have a negative impact on nutrition. 3. Isoflavones Soybeans contain many aromatic compounds, flavones and isoflavones, distributed in varying concentrations throughout the plant (Graham 1990). The quantity of these compounds varies depending upon growing conditions, 19 location, and variety of soybean (Eldridge & Kwolek 1983). These are thought to protect the plant from infection, regulate cellular processes, and also aid in the symbiotic process of nitrogen fixation (Sadowsky et al. 1991, Parniske et al. 1991). Genistin and its aglycone genistein account for the majority of the isoflavones found in soybeans with ~2-3 mg/g (Eldridge & Kwolek 1983). The isoflavones remain intact, to a large degree, upon processing of soybean into soy products such as flour, tofu, soymilk and miso (Murphy & Wang 1993). Substantial quantities of these isoflavones have been identified in the urine of primates (Adlercruetz et al. 1986) and humans (Adlercruetz et al. 1991, Axelson et al. 1984) consuming soy products. Of the isoflavones most research has focused on genistein which has demonstrated the most promise in inhibiting tumorigenesis. B. Genistein Genistein is an isoflavone with a molecular weight of 270.2 and a melting point of 297:8W: (The Merck Index 1989). It is insoluble in water and oil but freely soluble in alcohol. Genistein and its glucose conjugate genistin are found in many plants, particularly soybeans where they are found at concentrations of 1-3 mg/g (Eldridge & Kwolek 1983). Genistein has demonstrated estrogenic effects 20 (Carter et a1. 1955), antioxidant activity (Wei et a1. 1995), topoisomerase II inhibition (Markovits et al. 1989), and tyrosine kinase inhibition (Akiyama et al. 1987). 1. Estrogenic Activity of Genistein Phytoestrogens are a group of compounds found in plants that are capable of producing estrogenic effects. Some of the isoflavones in soybeans, genistein in particular, are also referred to as phytoestrogens due to their ability to mimic the effects produced by estrogen, albeit requiring much greater concentrations (Carter et al. 1955). Estrogens and estrogenic compounds such as the phytoestrogens elicit their effects through binding to and transforming estrogen receptors (ER) to a DNA binding form which binds to estrogen response elements leading to transcription of the estrogen responsive gene (Hirst et al. 1992, Reese & Katzenellenbogen 1991). Early assays assessed the estrogenic potency of compounds by monitoring the increase in mouse uterine weight upon feeding or injecting the compound (Farmakalidis et al. 1985, Folman & Pope 1966). Current assays utilize molecular approaches to assess estrogenic activity of a compound. For example, competitive binding studies quantitate the affinity of compounds, relative to estradiol, to the estrogen receptor. Genistein's affinity for the estrogen receptor is 21 reported to range from 1/50 to 1/1000 that of estradiol depending upon the species (Verdeal et al. 1980, Martin et al. 1978, Shutt & Cox 1972). Other assays assess the estrogenic effects of compounds by measuring their ability to increase proliferation of estrogen responsive cells (Makela et al. 1994), increase chloramphenicol acetyl transferase (CAT) expression of estrogen receptor-CAT constructs (Miksicek 1994), or expression of estrogen regulated proteins (Mayr et al. 1992). a. Uterotrophic Effects of Genistein The consumption of certain plants and plant products can result in impaired reproductive function in livestock. Over five decades ago a syndrome (clover disease), with effects ranging from temporary to permanent infertility, was described in sheep foraging upon subterranean clover in western Australia (Bennets et al. 1946). Additional studies have documented impaired reproductive function in a number of species (reviewed in Price & Fenwick 1985) including desert quail feeding upon desert brush (Leopold et al. 1976). Furthermore, a decrease in reproductive performance was observed in female rats fed either a soy-based diet or a diet supplemented with genistin (Carter et a1. 1955), and in male mice fed genistin (Matrone et al. 1955). All of these effects were attributed to the phytoestrogen content of the 22 diets. Phytoestrogens include the isoflavones, lignans, and other non—steroidal chemicals found in plants and plant products. These compounds can bind to the estrogen receptor and are thought to elicit their estrogenic effects through mechanisms similar to that of estradiol. It was later discovered that genistein was responsible for the impaired reproductive performance seen in sheep ingesting subterranean clover (Bradbury & White 1951). Genistein is an isoflavone (4',5,7—trihydroxyisoflavone), which has estrogenic activity (Folman & Pope 1966), and is present in various plants including legumes (Naim 1974). Since the initial discovery of its estrogenic activity there has been a number of studies in which the effects of soy and genistein upon the uterus of mice and rats were evaluated (Farmakalidis et al. 1985, Carter 1953): all of these have demonstrated estrogenic effects except the work of Farmakalidis & Murphy (1984). In their study the potent estrogen agonist diethylstilbestrol also did not promote uterotrophic effects in this strain of mouse (CD-l). b. Mammatrophic Effects of Genistein Mammary development in the rat is controlled by estrogen, progesterone, growth hormone and prolactin (reviewed in Topper & Freeman 1980). Estrogen acts directly 23 at the mammary gland by inducing gene transcription and the subsequent synthesis of many proteins, including the progesterone receptor (Horwitz & McGuire 1978). Estrogen also acts indirectly through the induced synthesis and release of prolactin from the anterior pituitary gland which then elicits its mitogenic effects on the mammary gland (Jones & Naftolin 1990). Prolactin synthesis in the anterior pituitary gland is under tonic inhibition by dopamine produced in the hypothalamus (Jones & Naftolin 1990). Estrogen is thought to decrease the activity of tyrosine hydroxylase, thereby decreasing the concentration of dopamine in the hypothalamus and pituitary gland (Jones & Naftolin 1990) resulting in increased plasma prolactin. Removal of endogenous estrogen will result in regression of the mammary gland, particularly the lobulo-alveolar structures. The estrogenic activity of genistein could affect the mammary gland in a manner similar to that of estradiol. There have not been any reports on the estrogenic effects of dietary genistein upon the mammary gland. However, there are recent data that show that genistein, when subcutaneously injected on days 23, 25, 27, and 29 postpartum into immature rats, will promote mammary gland development (Brown & Lamartiniere 1995). In this study 24 genistein increased cellular proliferation in the terminal endbuds, increased the number of Type I lobules (differentiated endbuds) and increased mammary gland size. c. Antiestrogenic Effect of Genistein The estrogenic effects of genistein upon the uterus are well known. However, genistein also has demonstrated antiestrogenic activity. When genistein was coadministered with estrone, genistein caused a suppression in the uterine weight increase seen with estrone alone, suggesting antiestrogenic effects (Folman & Pope 1966). These contradictory effects are not unusual for a compound. The chemical primarily used in the treatment of estrogen receptor positive breast cancer, tamoxifen, which is believed to compete with estrogen for the estrogen receptor, also has estrogenic properties when given to ovariectomized rats (Nicholson et al. 1988, Galman et al. 1990, Powers et a1. 1989, Martinez-Campos 1986). Like tamoxifen, the estrogenic or antiestrogenic activity of genistein is dependent upon conditions in which it is used. For example, tamoxifen exerts uterotrophic effects in ovariectomized rats; however, in intact rodents tamoxifen does not appear to exert estrogenic effects but to the contrary can have antiestrogenic activity providing the dose is high enough. Genistein may also function in a similar manner. 25 2. Antioxidant Activity of Genistein Metabolic reactions often produce reactive oxidized chemicals that can damage cell membranes and cause mutations in DNA. Genistein functions as an antioxidant and may potentially inhibit free radical formation thereby reducing cellular damage. For example, genistein suppressed HJL formation by the tumor promoter 12-O-tetradecanoylphorbol— 13-acetate (TPA) in HL-60 leukemia cells (Wei et al. 1993). In these studies topical genistein also inhibited TPA induced HKL formation in mouse skin. Wei (1996) found that genistein inhibits oxidation of guanine residues in DNA. In this system 8-hydroxy-2'-deoxyguanosine formation (oxidized guanine) induced by UV light was inhibited when genistein was present in the reaction mixture. These experiments provide evidence that genistein may be able to inhibit oxygen radical formation and thereby decrease DNA damage. Genistein has also been shown to inhibit DNA adduct formation in mice treated with the mammary specific carcinogen DMBA (Giri & Lu 1995). 3. Genistein Inhibits Topoisomerase II Topoisomerase II (Topo II) activity is required during replication of DNA. Topo II catalyzes double strand breaks in the DNA allowing the DNA to unwind to facilitate replication of the genome. Genistein (36-100 uM) inhibits 26 DNA topoisomerase II decantenation activity and stimulates Topo II:DNA complex dependent strand breaks (Yamashita et al. 1991, Markovits et al. 1989, Constantinou et al. 1990, Kondo et al. 1991, Okura et al. 1988). Genistein is thought to act in stabilizing the Topo II:DNA complex causing strand breakage. It does not intercalate, rather it is thought to interfere with the binding of ATP to Topo II. Topo II inhibitors are known to cause cells to differentiate (Constantinou et al. 1990). This has been observed in various cell lines and is confirmed by morphological changes occurring in the cell along with a change in proteins synthesized to those of a differentiated cell. Cell differentiation has also been demonstrated in cells treated with genistein. Whether cell differentiation is due to Topo II inhibition or tyrosine kinase inhibition is not clear. Some researchers have suggested that inhibition of tyrosine kinase activity was responsible for inducing cell differentiation (Rocchi et al. 1995, Watanabe et al. 1991, Honma et al. 1991). In the study by Honma (1991) genistein induced differentiation of cells transfected with v-abl (Honma et al. 1991). V-abl is thought to block differentiation of these cells. It was thought that genistein (18 uM) caused cell differentiation by blocking phosphorylation of v-abl on tyrosine residues thereby 27 inhibiting v-abl’s kinase activity. The later studies did not look at Topo II activity nor did the earlier studies look at tyrosine kinase activity thus it is possible that genistein inhibited both activities leading to cell differentiation. 4. Genistein Inhibits Tyrosine Kinases Protein kinase activity is essential in all cells. The activity of enzymes involved in signal transduction pathways are controlled by phosphorylating serine, threonine and tyrosine residues in the protein. Genistein does not inhibit serine or threonine kinases however genistein is a non-specific inhibitor of tyrosine kinases (Akiyama et al. 1987). In_yi;;g studies have shown that genistein inhibits a number of tyrosine kinases with ICw's ranging from 1 uM to >100 uM (Akiyama et al. 1987, Geissler et al. 1990, Huang et al. 1992). Protein tyrosine phosphorylation occurs during mitogenic activation of cell membranous receptors and the subsequent activation of cytosolic kinases involved in the regulation of proliferation and control of the cell cycle in a variety of cell types. As a tyrosine kinase inhibitor genistein may interfere with the phosphorylation of tyrosine residues on various proteins, particularly those that function in signal transduction pathways, leading to changes in the activity of these proteins. Inhibition of 28 tyrosine kinase activity would result in changes in the regulation of cell proliferation and may play an important role in the prevention and treatment of cancer (Akiyama et al. 1987, Nakafutu et al. 1992). For example, genistein was conjugated to an antibody developed against the CD19 antigen found on leukemia cells. Treatment of mice with 25 ug of the immunocomplex eliminated 99.999% of the leukemia cells whereas the antibody by itself, 50 ug, had no effect (Uckun et al. 1995). It is thought the immunocomplex targeted genistein to the CD19 receptor causing inhibition of src tyrosine kinase activity associated with and induced through the CD19 receptor, thereby leading to death of the leukemia cells. Inhibition of tyrosine kinases may therefore have profound effects upon the proliferation of tumor cells. C. Breast Cancer Etiology Normal functioning cells are usually in a quiescent state of the cell cycle called gap zero (G0). Stimulation of the cell causes entry into gap 1 UL) phase where the necessary proteins required in the replication of DNA (S phase) are synthesized. In late Gl there is a checkpoint, between Gltand S, called the restriction point where the cell decides whether conditions are appropriate to enter S phase and replicate the DNA. If conditions are not 29 satisfactory the cell will arrest in Glior S phase until conditions are corrected and then proceed. During S phase and into gap 2 (65) phase the cell monitors the progress of DNA synthesis and if all is well will enter into mitosis (M) phase where the cell divides. Cell division will not occur until the genome has been properly replicated and the necessary machinery required for mitosis is present. If this is not the case then the cell will arrest at Gzcn:bd phase. Tumorigenesis occurs through stages in which a normal cell is transformed into an immortal cell. These stages are initiation in which the DNA is mutated, promotion in which the growth of the mutated cell is favored, and progression in which the cells acquire the ability for anchorage independent replication and growth. The following section briefly describes the role that genistein could play in initiation and progression while genistein’s role in promotion is covered in much more detail. This is because the tumor model employed in my studies uses cells that are already tumorigenic and these cells do not progress to metastasis. 30 1. Genistein and Initiation: Initiation can be defined as a mutation in the genome that when not corrected leads to a change in function or activity of the gene product that will confer a growth advantage to the cell. Initiation can result from autosomal or somatic mutations. Autosomal (inherited) mutations are responsible for a small number of cancers and at the present time cannot be prevented; however, detection and interventions can be implemented to delay or prevent the onset of tumorigenesis (Henderson 1993, Henderson et al. 1993). For example women with a family history of breast cancer due to the inherited defective BRCAl gene may undergo mastectomy to prevent the development of breast cancer (Roberts 1993). Somatic DNA mutations, on the other hand, can be prevented given the agent responsible for the insult can be identified and avoided in the future. This implies somatic mutations occur from exposure to an environmental mutagen, an initiator. This is true in a large number of cancers; however, inherent errors in the replication of DNA account for some. During the normal cell cycle, errors in DNA replication with or without external stimulus, commonly occur but are usually corrected before or during the DNA synthesis phase (S-phase) of the cell cycle (Al-Khodairy & Carr 1992, Rowley et al. 1992). However, at times mutations 31 are not corrected and if these mutations occur in a gene whose product is responsible for regulation of cell proliferation, uncontrolled cellular proliferation may occur resulting in tumor formation. For example a mutation could result in the synthesis of a dysfunctional protein whose normal function is regulation of the cell cycle. The presence of this protein could disrupt timing of the normal cell cycle, decreasing the time allowed for DNA repair, resulting in the incorporation of a number of additional mutations in the genome. It is thought that the initial mutation infers a growth advantage to the cell, perhaps less responsive to negative growth factors, although at this point rarely is the cell transformed to a tumorigenic cell. However, promotion of this growth advantage causes instability in the genome making the cell prone to additional mutations which can eventually lead to a tumorigenic cell (Kaufmann & Kaufman 1993). For example mutations in the tumor suppressor gene p53 eliminate a checkpoint in the cell cycle. Normally p53 is induced following DNA damage which causes cell cycle arrest at Gg/S or G2. Cell cycle arrest provides the cell with an opportunity to correct the damage. By eliminating this checkpoint cells are likely to replicate the mutated DNA potentially contributing to greater instability (chromosomal 32 rearrangement, gene amplification) in the genome (Hartwell 1992). Cell specific mitogens are initially required to stimulate (promote) normal cells from quiescent CH phase to enter and progress through G1 phase, afterwards they are not required for progression of the cell cycle through 3, C5 and M phases. Mutations (initiation events) occurring in genes that encode proteins involved in normal cell growth could result in the synthesis of a dysfunctional protein that could cause uncontrolled cell growth. For example a point mutation in the gene encoding the proto oncogene p21ras (a cytosolic tyrosine kinase) causes the protein to become oncogenic (constitutively active). p21‘as is involved in signal transduction pathways (described below) that eventually culminate in cell proliferation. Tyrosine kinase activity is required for cell growth, thus a cell with mutated p21“as may acquire a growth advantage due to constitutively active tyrosine kinase activity. Studies have found that p21““ is mutated in rats treated with the mammary carcinogen DMBA (Zarbl et al. 1985). Genistein’s ability to inhibit DNA adduct formation in mice treated with DMBA (Giri & Lu 1995) suggests genistein could prevent DNA damage and therefore possibly prevent the initial stages of tumorigenesis. Mutated p21ras is found in the MDA-MB—231 33 human breast cancer cell line (Kozma et al. 1987). Mutations in p21““ are uncommon in human breast cancers however mutations in the tumor suppressors p53 and Rb tumor suppressor proteins are common. For example, analyzes of breast tumor tissue have found that p53 is mutated in 61% and Rb in 35% of the samples (Yokota & Sugimura 1993). Whether genistein is capable of preventing mutations in these and other proteins is unknown. 2. Genistein and Promotion: Initiated cells are still dependent on growth factors or other stimuli for proliferation, or require these factors to promote proliferation of the initiated cell. Promotion of genetically unstable initiated cells can lead to additional mutations in negative growth regulator (tumor suppressor) or positive growth regulator (proto oncogenes) genes and result in a tumorigenic cell that is no longer dependent upon growth factors or promoters for proliferation. For example, estradiol is thought to be required initially in mammary tumorigenesis and these tumors have a better prognosis; however, over time these cancers progress to an estrogen independent state associated with a very poor prognosis. The mechanisms responsible for a tumor progressing to hormone independence are not clear but could involve synthesis of a dysfunctional protein in the 34 signaling pathway of estradiol allowing growth independent of estradiol. Estradiol functions as a mitogen in part by binding to the ER and inducing expression of several genes including the progesterone receptor (Horwitz & McGuire 1978) and transforming growth factor alpha (TGF-d)(Saeki et al. 1991). TGF-d binds to the epidermal growth factor receptor (EGFR) resulting in autophosphorylation of the EGFR on tyrosine residues which then serve as substrate for the binding of cytosolic proteins containing the src homology domain 2 (SH2) (Sierke & Koland 1993, Koch et al. 1991, Russell et al. 1992). This serves to localize the targets of EGFR to the EGFR where they are then activated by phosphorylation on tyrosine residues and eventually results in cell division (Zhu et al. 1992, Feng et al. 1993, Gale et al. 1993, Zhu et al. 1993). p21“”, a proto-oncogene, is one protein that interacts with the EGFR (Gale et al. 1993, Li et al. 1993). Activated p21.“as phosphorylates raf, a cytosolic kinase, on a tyrosine residue and activates it. Raf then phosphorylates and activates cytosolic mitogen activated protein kinases (MAPKK) which then phosphorylate and activate MAPK’s p42MAPK and p44W”K(Kyriakis et al. 1992). Nuclear p42/44’WK phosphorylates a number of nuclear proteins including the transcription factors c-myc and c-jun (Pulverer et al. 35 1991). This causes the formation of active transcription complexes that initiate transcription of early genes whose products are necessary for cell cycle progression. One of the early genes required for entry into and progression through G1 is cyclin D1 (Sherr 1993). Cyclins bind to and activate constitutively expressed kinases (cdk’s). Cyclin D1 binds to cdk4 and cdk6 and, as the levels of cyclin D increase, activates the kinase activity of the cyclin D:cdk4 complex. One of the key proteins phosphorylated by cyclin D:cdk4 is the key negative growth regulator protein retinoblastoma (Rb). Rb, in the hypophoshorylated state, binds to the transcription factor E2F and prevents E2F from initiating transcription of early genes required for G1 (reviewed in Weinberg 1995). Hyperphosphorylation of Rb by cyclin D:cdk4 causes dissociation of E2F and allows E2F to initiate transcription of genes required for G1 to S transition. After the cells have passed through Gl'phase they will normally complete mitosis. Mitosis occurs concurrently with the activity of M— phase promoting factor (MPF), a serine/threonine kinase, suggesting MPF is required for mitosis. MPF is composed of cyclin B and p343x2. :U1C% cyclin B synthesis increases and forms a complex with the dephosphorylated form of p34““2. The activity of the cyclin B:p3¢”” complex is then 36 controlled by phosphorylation and dephosphorylation of cdk p34md, the catalytic unit (King et al. 1994). If the intracellular conditions required for mitosis (intact duplicated DNA, cytoskeletal structure, proper cell mass) are met then mitosis will proceed. The controlled activity of tyrosine kinases involved in signal transduction pathways is essential for normal cell proliferation. Uncontrolled activity of certain tyrosine kinases can produce a cell that is unresponsive to negative growth factors that normally suppress cell growth. In fact tyrosine kinase activity is increased in human breast cancer (Hennipman et al. 1989, Bolla et al. 1993, Lower et al. 1993). In addition many of the oncogenes identified thus far are tyrosine kinases (Smith et al. 1993). As a tyrosine kinase inhibitor genistein could potentially affect a number of tyrosine kinases involved in signal transduction pathways. For example, amplified or mutated p185mu, an EGFR like membrane bound receptor with tyrosine kinase activity (Stern et al. 1986, Wildenhain et al. 1990), is present in 20% of human breast cancers (Borg et al. 1991). p185neu functions in a manner similar to that of the EGFR (discussed above) in that it activates p21“as (Ben—Levy et al. 1994) and initiates a signal transduction pathway involving raf and the MAPKK cascade, eventually resulting in cell 37 proliferation. Whether genistein can inhibit pl85“”.as it does the EGFR (1 uM) (Akiyama et al. 1987) is unknown; however, as a tyrosine kinase inhibitor genistein could play an important role in inhibiting the promotion of initiated cells. Genistein has also been shown to inhibit GDP GTP exchange on p21“” by inhibiting EGFR phosphorylation. Inhibition of EGFR induced responses can occur at the receptor or downstream of the receptor. For example the activation and activity of MAPK in stimulated human neutrophils is inhibited by 36 uM genistein (Torres et al. 1993). Genistein competes with ATP for the ATP binding site in tyrosine kinases (Akiyama et al. 1987). The ATP binding site domain is well conserved among tyrosine kinases thus genistein has the potential to inhibit many other unidentified tyrosine kinases, potentially functioning in control of cell growth, in addition to those described above (Hanks et al. 1988). Genistein, in addition to inhibiting the tyrosine kinase activity of the EGFR, inhibits the oncogenes v-abl (39 uM)(Geissler et al. 1990), pp60""src (26 uM), and ppllOgaq"fes (24 uM). Genistein also inhibits fibroblast growth factor (FGF) and insulin induced p21“”:GTP complex formation in rat pheochromocytoma cells (PC- 12)(Nakafutu et al. 1992), and erythropoietin induced p21““:GTP formation in human erythroleukemia cells (Torti et 38 al. 1992). In addition to genistein's ability to inhibit tyrosine kinase activity genistein also inhibits Topo II activity (discussed above). Many cancer chemotherapeutic drugs aCt by inhibiting topoisomerase II (Topo II) activity (Corbett et al. 1993). These drugs are thought to act by stabilizing the Topo II:DNA complex, by inhibiting Topo II's catalytic activity, leading to strand breakage and cell cycle arrest at Gpflu (Ishida et al. 1991). Genistein’s inhibition of Topo II activity could therefore also inhibit promotion of initiated cells. 3. Genistein and Progression: The third stage of carcinogenesis is progression. After the cells have been initiated and the growth of initiated cells promoted, the tumorous cells can progress to an invasive and sometimes metastatic state. These cells are characterized by a number of mutations in addition to changes in cellular activities, in particular the extent of vascularization (Marx 1993). The density of vessels in the tumor is highly predictive of the potential for metastasis. A vasculature must be established in order for a solid tumor mass to grow beyond 2-3 mm? in diameter (Folkman 1989). Genistein has been shown to inhibit angiogenesis in_xitrg (150 uM) and has also been shown to inhibit endothelial cell 39 proliferation (5 uM), thus genistein could prevent progression by inhibiting angiogenesis (Fotsis et al. 1993). In addition, genistein (1 uM) has been shown to inhibit the in_yitrg invasion of a highly metastatic murine mammary tumor cell line (Scholar & Toews 1994). D. Antiproliferative Effects of Genistein: If genistein is capable of inhibiting tumor cell growth in vitrg then it may be able to inhibit tumor cell growth in 111g. Genistein has been shown to inhibit proliferation of both estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-468) human breast cancer cells in 21:29 (Peterson & Barnes 1991). Recent studies have also shown genistein will inhibit the growth of estrogen dependent MCF-7 adriamycin resistant cells and MDA-MB-231 estrogen independent human breast cancer cells with an IC50 of 7.74 uM and 15.02 uM respectively (Monti & Sinha 1994). Furthermore, genistein inhibited epidermal growth factor (EGF) stimulated AGS human gastric cancer cells with an IC50 of 7 uM (Piontek et al. 1993). Genistein has been shown to reversibly arrest cell cycle progression of human gastric cancer (HGC-27) cells at Cfi/M (Matsukawa et al. 1993). Genistein also arrests the cell cycle at GQ/M in Jurkat T-leukemia cells while at higher doses blocked cell cycle progression through 5 phase 40 (Spinozzi et al. 1994). In this study genistein also induced apoptotic cell death. The phosphorylation of tyrosine residues in key cell cycle regulatory proteins control the progression of the cell through the cell cycle. The inhibition of tyrosine kinases by genistein may therefore be responsible for the observed effects on cell proliferation and the cell cycle. For example inhibiting tyrosine kinases involved in early Gloooe :o\o o.m m HN~.H H m.m. Hs.m H H.s. H.o m Her.m H m.m. so.r H m.o. 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Nboo\>< HoochH\mH3 mm (measured by caliper) at the time of grouping were used in the study (n=27). Tumor length (1) and width (w) were measured weekly for 5 weeks and cross- sectional area determined by the following formula (l*w/2) n. Food intake was measured during the last 3 days of the study. At the end of the study mice were anesthetized, weighed, bled via cardiac puncture, and killed by overexposure to anesthesia. Genistein did not affect the growth of tumors in the previous study so the amount of genistein fed to mice was increased to 3000 pg/g AIN-93G. Female athymic mice (n=27) were received at four to five weeks of age, sorted 3 or 4 mice to a cage and allowed unrestricted access to water and AIN-93G diet. One week later the mice were injected subcutaneously with MDA-MB-231 human breast cancer cells (as described above). After five weeks the mice were grouped, to equalize tumor number and cross-sectional area, into a 3000 pg/g genistein/AIN-9BG group (n=l4) and an AIN-93G control group (n=l3), and treatments began. Tumor measurements and analysis were performed as described above. To evaluate the effect of dietary genistein on the initial formation and growth of tumors the following study was conducted. Female athymic mice (n=30) were received at 3-4 weeks of age and allowed unrestricted access to water 92 and the AIN-93G diet for 7 days. One group of mice (n=15) were fed AIN-93G diet supplemented with 750 pg genistein/g AIN-93G, 3 days prior to injecting the MDA-MB-231 cells. Inoculation of tumor cells and tumor measurements were performed as described above. e. High Pressure Liquid Chromatography Analysis (HPLC) of Plasma Genistein: Mice were anesthetized and bled by cardiac puncture. Blood (approximately 800 pl/mouse) was placed in microfuge tubes containing 10 pl 15% EDTA, centrifuged at 12,000 x g and the plasma removed and stored at -20W3. To determine conjugated and unconjugated genistein 50 pl of plasma was aliquotted in duplicate and one set received 5 pl (515 units) of B-glucoronidase Type H- 1 (Sigma, St. Louis, MO.). All aliquots were incubated in 0.5 ml microfuge tubes at 37°C for 24 h. Following the incubation, 50 pl of absolute methanol was added to each tube, the tubes vortexed and then centrifuged at 15,000 x g for 10 min. Approximately 75 pl was removed and placed at - 20%: until analysis. For analysis of genistein the microfuge tubes were centrifuged at 15,000 x g for 10 min. and 20 pl injected onto a C18 column (Microsorb-MV, 5 pM 100A, Rainin Instrument in Woburn, MA) with a flow rate of 1.0 ml/min of 50:50 methanol:water with 0.1% acetic acid. 93 Recovery was determined by adding a known amount of genistein (28 pl of 21.1 pg/ml in ethanol) into 600 ul plasma from control mice and diluting this with plasma to a final concentration of 1.82 pM genistein. Mean recoveries were determined to be 96 i5.87%. The data presented are not corrected for recovery. Genistein was not detected in the plasma of control mice fed the AIN-936 diet. To avoid artificially manipulating the data obtained from HPLC analysis of plasma genistein, mean plasma genistein concentrations are reported only for samples in which genistein was detected. The level of detection for plasma genistein was ca. 130 nM. f. Statistical Analyzes: All statistical tests were performed using a PC—based version of the Statistical Program for the Social Sciences (SPSS) Version SPSS/PC 7.5, Chicago, IL 60611. At the end of each experiment tumor area per mouse, food intake per cage and mouse weight were analyzed by student’s T-test. Values in the text and figures are means i SEM. 94 ”0:" _I__I_ .- OuM 10uM 2011M 40uM 8011M Genistein Concentration Figure 4. Effect of Genistein on MDA-MB-23l Cell Proliferation. MDA-MB-231 human breast cancer cells were treated with 0, 10, 20, 40 and 80 pM genistein in MEM containing 10% FBS for a period of 7 days. Fluorometric analysis of cellular DNA was performed on days three and seven. Solid bars represent cells cultured for three days and open bars are data collected after seven days. Each point represents the mean i SEM of eight measurements. 95 D. Results 1. In Vitro MDA—MB-231 Cell Proliferation and Cell Cycle Analysis: In order to assess the effect of genistein on cell proliferation the following studies were conducted. Genistein inhibited proliferation of MDA-MB-231 cells with an ICSC of approximately 20 pM (Figure 4). Genistein at 10 pM had no effect on cell proliferation whereas 20, 40 and 80 pM resulted in a progressive decline in proliferation. To evaluate a potential mechanism for growth inhibition studies were conducted to determine whether the inhibition of cell proliferation by genistein resulted from perturbations in the cell cycle or perhaps cytotoxicity. Genistein at 40 and 80 pM produced a marked block in the Ch/M phase of the cell cycle (Figure 5 and Table 5). Cell viability, as assessed by the cellular exclusion of trypan blue dye, was greater than 86% at all concentrations of genistein. Viability was actually greater than 93% at all time points with the exception of 48 hours where samples treated with 40 and 80 pM genistein had mean viabilities of 86%. 2. Dose Response Study of Dietary Genistein on Plasma Genistein and Food Intake: The concentration of genistein required to inhibit MDA-MB-231 cell proliferation in_xitrg HDVHO w. Mmmmon 0m omswmnmws cpo: nvm nmpw ouzwume Ompwm. noHH nwowo vbnwwuwn "aflawunowa_ u! 0 we no .0 no a»... an? a. a 0.x: 9 m 9.). a. m a»). a. a 0.x: 9 a 0.? o mo mm H» an up be up mm He no as pm pm am pm we mm H. MN n. .H on mm um us mm um a. No .0 up be mm as up .o an on m me No on as um NH on mg no No as r. 4» mm up as am am am am Am Hw wH up uw Hg up mu * Umnm Hm mxpnmmwmd mm m pmnomsn 0m nzm ompwm H: moo: promo 0m n30 ompp oIZmINwH omppm zmnm nnmmnma sen: Ho. mo. so man mo :3 omzwmnmwo M: 2m: noonmwswoo How mwm mom m UmHHOQ om H». N». am mad om :ocnm. nmppm smnm mnmwsmd awn: pmopwowca woawam mad momw<~ma U< mp0: o~H HZMZH 3x. mum + u: :3 Caz 33 Hum + .3 ES nmz ( 0. Q. .. 3.x. .. _, m ox: It; :.\. 52> OOZHMZH 39.8 m .\.. mum + a: :7— Caz Am :2 uwra 99 was approximately 20 pM. Whether a similar plasma concentration can be produced in mice fed genistein is unknown. To determine the plasma concentration of genistein produced by feeding different amounts of genistein, Balb/C mice were fed genistein at doses ranging from 0—6000 pg/g AIN-936. The plasma concentration of genistein (free + conjugated) in mice fed genistein at doses from 0 to 6000 pg/g AIN-93G ranged from 0 to 7.1 i 1.25 pM (Figure 6). The plasma concentration of unconjugated genistein ranged from 0 to 1.76 i 0.339 pM. To assess whether genistein would alter food consumption, groups of mice were fed the control diet (AIN- 936) for 4 days and then switched to diets containing from 0-6000 pg genistein/g AIN-93G for 4 days. Food intake was assessed each day. Food intake did not differ with dietary genistein at any of the doses tested (data not shown). 3. Effect of Genistein Fed to Mice Inoculated with MDA-MB-231 Cells: To determine whether the observed antiproliferative effects of genistein in cell culture could also occur in yin the following studies were conducted. 100 E! :3 .E9fi0 : .2 3720— L: H 5 :540- C) U .5350- G) H .23 5130~ CD ii 63 E _. I: ii m0.00 5 0 375 750 1500 3000 6000 a. Genistein Content of Diet in ug/g Figure 6. Plasma Genistein Concentration in Mice Fed Genistein. Balb/C mice were received at 4 weeks of age, sorted to equalize weights (3 mice/cage, 2 cages/group, n=6), and fed AIN—93G (6 g/d) for two weeks. Mice were then given 6 g of AIN-936 per day without genistein for four days and then switched to AIN-93G diet containing genistein at the doses noted above for four days. Blood was collected, plasma prepared for analysis of genistein content by reverse phase HPLC. Dark bars represent total genistein (free + conjugate) and open bars represent free genistein. Values are expressed as mean i SEM. 10] Dietary genistein was fed at a dose (750 pg genistein/g AIN- 936) which produced a plasma genistein concentration of approximately 1 pM. Xu et al. (1994) have found total plasma genistein concentrations of approximately 1 pM in women consuming soymilk which contained 36 mg of genistein. This dose also exerted biological activity in estrogen responsive tissues in mice. This dose of dietary genistein did not significantly affect the growth of solid tumors (Figure 7). Plasma genistein (free + conjugated) was 0.933 i 0.107 pM and the free form was 0.306 t 0.141 pM. A plasma concentration of approximately 1 pM genistein appears to be insufficient to inhibit the growth of solid tumor masses. To determine the effect of a higher dose of genistein on tumor growth, genistein was fed at 3000 pg/g AIN-936. This lead to an increase plasma genistein concentrations (5.88 pM i 0.56, conjugated + free form, and 0.64 pM i 0.18 free form) and allowed us to assess the effect a larger dose of genistein would have on tumor growth. Tumors in the group of mice fed 3000 pg genistein/g AIN-93G grew significantly slower and at the end of five weeks were 23% smaller than tumors in the control group (Figure 8). 102 200 N 160 ” E E F JD :5: 120 — ' m I- ‘< I! E E3 80* CONTROL E n=33 {in} 2 40 _ cells “$6th treatment started 0 l 1 1 1 -l 0 5 6 7 8 9 10 Figure 7. Tumor Growth in Mice Fed 750 pg/g Genistein After Tumor Formation. MDA-MB-23l cells were inoculated into the mice as described in the materials and methods. Mice were grouped to equalize tumor number and size into AIN-93G and AIN-93G plus genistein. The solid line represents mice (n=13) fed dietary genistein (750 ug/g) and the dashed line represent control-fed mice (n=l4). Tumor growth was measured weekly. Values represent mean tumor area/mouse i SEM. 103 Food intake per cage was 10.7% (p50.001) lower in the genistein fed mice compared to control mice (13.24 i 0.19 vs. 14.83 i 0.22). Weight gain, although lower in the genistein fed mice (2.06 i 0.33 g vs. 2.69 i 0.59 g), did not differ significantly (p=0.36) between the groups. Dietary genistein (750 pg genistein/g AIN-93G) did not significantly affect tumor growth (Figure 7) when given after the formation of solid tumor masses. Development of a vasculature is required for solid tumor growth in excess of 2—3 mm (Folkman 1989). Genistein has been shown to inhibit the process of angiogenesis thus genistein may inhibit the initial development of a tumor (Fotsis et al. 1993). To assess the effect of genistein upon initial tumor development 750 pg genistein/g AIN-93G was fed three days before the MDA-MB-231 cells were inoculated into the athymic mice and continued throughout the experiment. Dietary genistein (750 pg genistein/g AIN-93G) did not significantly affect the development (number of tumors per group) or growth of tumors (Figure 9). In both of the studies where mice were fed 750 pg genistein/g AIN-93G, food intake or weight gain did not significantly differ between the two groups (data not shown). 104 2M) p—e a G I 120* Q Q I GENISTEIN n = 50 MEAN TUMOR AREA IN mm2 A c 1 cells Injected treatment began Figure 8. Tumor Growth in Mice Fed 3000 pg/g Genistein After Tumor Formation. MDA-MB-231 cells were inoculated into the mice. Mice were sorted to equalize tumor number and size into AIN-936 and AIN-936 plus genistein groups. Tumor growth was measured weekly. The dashed line represents mice (n=l4) fed dietary genistein (3,000 pg/g) and the solid line represents control-fed mice (n=13). Values represent mean tumor area/mouse i SEM. 105 150 ~ 8 '5 l I MEAN TUMOR AREA IN mm2 o~ c» l p— O) G treamtent started cells injected 0 ‘ L 1 1 L 1 -l 0 3 4 5 6 7 8 Figure 9. Tumor Growth in Mice Fed 750 pg/g Genistein Before Tumor Formation. Mice received dietary genistein three days before cell inoculation. MDA-MB-231 cells were inoculated into the mice and tumor growth measured weekly. The dashed line represents mice (n=10) fed dietary genistein (750 pg/g) and the solid line represents control-fed mice (n=9). Values in the graph represent mean tumor area/mouse i SEM. 106 E. Discussion We have demonstrated that genistein will inhibit the proliferation of MDA-MB-231 human breast cancer cells in 311:9 (Figure 4) and that the probable mechanism is inhibition of the cell cycle at GQ/M (Figure 5 and Table 5). These data are in agreement with the work of Peterson & Barnes (1991) using MDA-468 and MCF-7 cells and that of Monti & Sinha (1994) using MCF-7/WT, MCF-7/ADR and MDA-231 human breast cancer cells. In these studies the concentration of genistein required to inhibit the proliferation of human breast cancer cells by 50% ranged from 7 pM to 40 pM. In addition, others have shown that similar concentrations of genistein (7 pM to 68 pM) will inhibit the proliferation of various cell lines (Yanagihara et al. 1993, Piontek et al. 1993). In the present studies 40 pM and 80 pM genistein blocked cells in the GQ/M phase of the cell cycle. Hunakova et al. (1994) conducted studies with leukemic K—562 cells and demonstrated that 10 pM genistein produced a block at Gb/M. Matsukawa et al. (1993) treated HGC-27 gastric cells with 25 pM to 60 pM genistein and found a progressive block in GyH4. Traganos (1992) however, has shown that 74 pM genistein will block lymphocytic leukemia MOLT-4 in S phase in addition to a Gyfld block with 18.5 to 74 pM genistein. Spinozzi (1994) has 107 also demonstrated that 18.5 to 37 pM genistein will block Jurkat T-leukemia cells in.Gb/M while 74 to 111 pM genistein will block the cell cycle in S phase. It is likely that genistein is inhibiting one or more tyrosine kinases required in the transition through different phases of the cell cycle and that the perturbation in the cell cycle produced by genistein depends on the specific cell line utilized. To date no studies have been reported in which dietary genistein was fed to tumor bearing mice. To assess the effect of genistein upon the proliferation of MDA-MB-231 tumors female athymic mice were fed 750 pg genistein/g AIN- 936 after solid tumor masses had formed. This dose was selected based on data from previous studies in which 750 pg genistein/g AIN-93G exerted biological effects in estrogen responsive tissues (Santell et al. 1997). In addition 750 pg genistein/g AIN-93G produces a plasma genistein concentration of approximately 1 pM in mice which is similar to that seen in humans consuming soymilk containing 36 mg of genistein (Xu et al. 1994). However, this plasma concentration of genistein is much less than that required to inhibit MDA-MB-231 cell proliferation in_yitrg (Figure 4). Direct comparisons from in_xitrg data to conditions in vin cannot be made with certainty due to a number of 108 factors, one of which is serum concentration. Fetal bovine serum is typically supplemented at 10% v/v to maintain MDA- MB-231 cells. Whether mitogens, such as growth factors and steroids, contained in this formula are representative of those occurring in_yiyg is unlikely. Treatment of the mice with this dose of genistein (750 pg genistein/g AIN-93G) did not significantly affect the growth of solid tumor masses. In order to assess the effect a higher plasma concentration of genistein would have on tumor growth the amount of genistein fed to mice was increased to 3000 pg/g AIN—93G. Results from feeding Balb/C mice different amounts of genistein, indicate that a total plasma genistein concentration of 3.3 pM could be achieved in mice fed 3000 pg genistein/g AIN-93G (Figure 6). There are data that indicate that human consumption of soymilk containing 226 mg genistein (total)/day will result in total plasma genistein concentrations of up to 6 pM (Xu et al. 1995). Thus, it is possible to achieve relatively high plasma concentrations of genistein in humans. Treatment of athymic mice with 3000 pg genistein/g AIN-936 significantly inhibited the growth of solid tumor masses when compared to control mice (Figure 8). Total plasma genistein concentration in the athymic mice fed 3000 pg genistein/g AIN-93G was 5.88 pM, 78% higher 109 than that in the Balb/C mice (3.31 pM) fed the same amount of genistein (3000 ug/g). The plasma concentration of free genistein was 0.64 pM in the athymic mice compared to 0.70 pM in the Balb/C mice. Whether the absence of microflora in the sterile gut of the athymic mouse is responsible for difference in total plasma genistein concentration, compared to Balb/C mice, is unknown. Food intake did not differ in the study in which genistein was fed to Balb/C mice from 0-6000 pg/g AIN-93G, however the mice were only fed for a period of 4 days. Caloric restriction has been shown to inhibit tumor formation and growth (Tannenbaum 1945) so food intake was monitored for 3 days in every week over the duration of the 5 week study. Food intake in the genistein treated mice was 10.7 % lower and this difference was significant; however, weight gain per mouse was not significantly different. Weight gain has a slightly higher correlation to tumor incidence than caloric intake although both weight gain and caloric intake are obviously related (Albanes 1987). In the previous study 750 pg genistein/g AIN-93G was administered to the mice after the formation of solid tumor masses. Solid tumor masses greater than 2-3 mm require a vasculature for nourishment. This vasculature is formed (angiogenesis) through an intricate balance of protease 110 activity resulting in degradation of the basement membrane in a controlled fashion thus permitting the generation and infiltration of blood vessels. Genistein has been shown to inhibit this process although the concentration required in 11119 was approximately 150 pM (Fotsis et al. 1993). This concentration is much greater than that observed in the feeding studies with genistein; however, the effect of lower concentrations of genistein upon the initial stages of solid tumor development in_1119 has not been investigated. In addition in_yitrg endothelial cell proliferation was inhibited with ICw's of 5 pM (Fotsis et al. 1993) and 12 pM genistein (Koroma et al. 1994). To assess the effect of genistein on initial tumor growth a study was conducted in which 750 pg genistein/g AIN-93G was given 3 days prior to inoculating the MDA-MB-231 cells. Genistein fed before inoculating the MDA—MB-231 cells and then continued throughout the experiment did not affect the appearance or growth of the resultant tumors (Figure 9). This is the first study that has demonstrated dietary genistein to inhibit the growth of MDA-MB-231 human breast cancer cells implanted into athymic mice. The amount of dietary genistein required to inhibit tumor growth was high (3000 pg/g) and would not be attained through a normal diet; however, as mentioned above it is possible to achieve plasma 111 genistein concentrations in humans (6 pM). Food intake was lower in the genistein fed mice and this could have contributed to the decrease in tumor growth in these mice; however weight gain was not different. This could be due to little variation in food intake within treatment groups and a wide variation in weight gain. The long-term physiological effects of a large dose of genistein (3000 pg genistein/g AIN-93G) are unknown. Genistein is a phytoestrogen and at the doses used in these studies does produce estrogenic responses in various organs, including the mammary gland, of ovariectomized rodents (Santell et al. 1997). Furthermore genistein has been shown to induce estrogen receptor positive human breast cancer (MCF-7) cell proliferation in_yitrg and (MCF-7) tumor growth in 1119 (Hsieh et al. 1996). The effect of administering genistein to post-menopausal women, particularly those susceptible to estrogen dependent cancers, is unknown. Before any recommendations can be made on the use of genistein as a prophylactic or treatment for estrogen receptor negative breast cancer, much more research is required. SUMMAR! AND FUTURE RESEARCH Summary The incidence of breast cancer is approximately 1 in 30 in Japanese women while it is approximately 1 in 8 in the US. As Japanese women migrate to the US there is an increase in breast cancer incidence in each of two subsequent generations approaching the incidence rate of breast cancer in the US. This suggests environmental changes, including dietary changes, are responsible for the increase in breast cancer. The diet of Japanese immigrants changes from one in which vegetables, including soy products, are largely consumed in Japan to one in which more meat and fat and less vegetables are consumed in the US. At the present time the role of fat in human mammary tumorigenesis is not clear. A number of prospective studies suggest that fat intake is not correlated with the incidence of breast cancer, however there are also a couple of studies that show a slight positive relationship. The consumption of soy products in Japan is many fold greater than that in the US. In addition Japanese immigrants to the US consume 112 113 less soy products than they do in Japan. Therefore the change in consumption of soy products could be one factor responsible for the changing breast cancer incidence pattern seen in Japanese immigrants to the US. Of the possible anticarcinogenic compounds in soy the one component that has demonstrated the greatest ability to inhibit cell growth in yitrg is the isoflavone genistein. I therefore conducted studies to assess the ability of this compound to inhibit breast cancer cell growth in_xitrg and in_yiyg. Genistein is a phytoestrogen and is capable of exerting estrogenic effects in the rodent uterus, however the biological effects of dietary genistein are not well characterized in other estrogen responsive organs. Therefore studies were conducted to assess the effect different doses of dietary genistein would have in the rodent by using estrogen responsive tissues such as the uterus, mammary gland and the hypothalamic/pituitary axis as indicators. In addition the plasma concentration of genistein responsible for these effects was determined. Dietary genistein (750 pg/g diet)increased uterine weight and uterine c—fos mRNA expression, stimulated lobular/alveolar development of the mammary gland and increased plasma prolactin. The plasma concentration of total genistein (free + conjugated) in rats fed 750 pg 114 genistein/g AIN-76A was 2.54 pM and the concentration of the free form was 0.4 pM. Competitive binding studies indicated that genistein competes with estradiol for the estrogen receptor with an affinity approximately l/lOOth that of estradiol. Genistein (750 pg/g AIN-76A) did not antagonize the action of concurrently fed estradiol (1.0 pg/g AIN-76A) in the organs studied in ovariectomized rats nor did dietary genistein (750 pg/g AIN-76A) inhibit the development of the mammary gland or uterus in immature intact rats fed genistein through sexual maturation (30 to 44 days of age). These studies have shown that 750 pg genistein/g diet is capable of exerting biological effects in 1119. They have also provided useful data that will help in deciding the initial dosage of genistein that will be used in conducting in vivg tumor studies. Antiproliferative effects of genistein have been observed in both estrogen dependent and independent cells in XLLLQ. I decided to conduct my in_vitrg and in 1119 studies using estrogen independent MDA-MB-231 human breast cancer cells. By using estrogen receptor negative cells the potential estrogenic activity of genistein in affecting estrogen dependent tumor formation and growth is eliminated. Genistein (20 pM) inhibited MDA-MB-231 cell proliferation in vitro by 50%. The cell cycle was blocked at GQ/M when 40 pM 115 or 80 pM genistein was added to the medium. To evaluate the effect of genistein on tumor growth in_xiyg, athymic mice were inoculated with MDA-MB-231 cells and fed (750 pg/g AIN- 93G) genistein. Genistein at this dose did not affect tumor growth. Next I conducted a dose response study to determine the plasma genistein concentration in Balb/C mice fed 0 to 6000 pg genistein/g AIN-93G. Genistein at 3000 pg genistein/g AIN—93G produced a plasma genistein concentration (free + conjugated) of 3.3 pM. When this dose was fed to mice an inhibition of tumor growth was observed when compared to untreated control mice; however, there was a 10.7% reduction in food intake in the genistein group. Although food intake was lower in the genistein treated mice compared to control mice there was no difference in weight gain. To assess the effect of dietary genistein on initial tumor development 750 pg genistein/g AIN-93G was fed before tumor cells were inoculated into the mice. This dose of genistein did not inhibit the development or growth of tumors. This research demonstrates the disparate characteristics of genistein. Genistein is mitogenic in estrogen responsive tissues in ovariectomized rodents yet is also antiproliferative in both estrogen dependent and independent cell lines in_yitrg. Other researchers have 116 demonstrated that low doses of genistein will stimulate the growth of estrogen dependent breast tumor cells in_yitrg and in yiyg; however, when higher doses of genistein are given estrogen dependent cell growth is inhibited in_yitrg. The stimulatory or inhibitory effect of genistein on cell growth are dependent on the dose. In the present studies 750 pg genistein/g diet did not inhibit tumor growth however tumor growth was inhibited with 3000 pg genistein/g diet. Food intake was lower in the genistein fed mice compared to the control mice and this could have contributed to the inhibition of tumor growth; however, weight change was not different between the two groups. The effects of dietary genistein upon development of mammary cancer in humans is unknown. These studies suggest genistein could either enhance (due to its estrogenic activity) or inhibit (due to its antiproliferative activity) mammary tumorigenesis. Additional research is needed on the effects of dietary genistein in tumor models that simulate tumor development in pre- and postmenopausal conditions. 117 Future Research These studies have answered some of the questions regarding genistein and its effect on breast cancer yet have also created additional ones. For example: 1) Relatively large doses of dietary genistein (750 pg/g) did not affect estrogen independent tumor growth yet did exert estrogenic effects in estrogen responsive organs in ovariectomized rodents. This suggests that this does of dietary genistein could potentially stimulate estrogen dependent breast cancer in ovariectomized rodents. Whether dietary genistein could have similar effects on the development of breast cancer in postmenopausal women is unknown yet clearly worthy of study in an appropriate animal model. The ovariectomized athymic mouse implanted with estrogen dependent breast cancer cells would be a useful model in which to conduct these studies. 2) Genistein did not antagonize estradiol in intact or estradiol supplemented ovariectomized rats. Nor was the estrogenic effect of genistein additive to that of estradiol. This suggests dietary genistein was not estrogenic, or the estrogenic activity was not measurable, in the presence of high levels of estradiol found in premenopausal women. Whether genistein would have a role in the promotion of breast cancer in premenopausal women is 118 unknown yet once again worth studying in the appropriate model. The intact female athymic mouse implanted with estrogen dependent human breast cancer cells would be a useful model in which to conduct this study. 3) A large dose of dietary genistein (3000 pg/g diet) did inhibit the growth of estrogen independent human breast cancer cells in the athymic mouse. However, food intake was lower in this group of mice compared to the control mice. Mouse weight did not differ between the two groups and weight is thought to be more highly correlated with breast cancer incidence. Nevertheless, it is unclear whether the decrease in food consumption could be partly responsible for the observed inhibition of tumor growth. This study should be repeated with the following conditions. Mice should be housed individually to enable a more accurate analysis of food intake per mouse. The amount of food provided to mice in the control and treatment groups should be slightly less than that consumed by the mice fed 3000 pg/g in this study. Furthermore a positive control group of mice provided with food ad lib should be included. This would eliminate the potential confounding variable of food intake upon tumor growth. 4) Genistein has been shown to inhibit both estrogen dependent and independent human breast cancer cells in 119 yitrg. Therefore the research described in number 3 should also be conducted utilizing estrogen dependent human breast cancer cells. Appendices 120 Introduction to tho Appendices The data presented in the attached appendices are from experiments that were not included in manuscripts or were from preliminary studies. These include a number of studies that evaluated the effect of administering genistein via different routes upon tumor growth, for example: gavaging genistein or implanting genistein pellets. Studies are presented that assessed the effect the gavage vehicle has on food intake. A study assessed the plasma genistein concentration over a 24 hour period after gavaging a genistein bolus to determine the timing of maximum plasma concentration. The eating behavior of mice was assessed to find out when they consume the greatest amount of food. These studies allowed us to determine what time to kill the mice in order to measure the highest plasma concentration that could be achieved from the diet. In addition the effect feeding different amounts of dietary genistein would have on food intake and weight gain was assessed. 121 Appendix A Effect of Genistein, with 1% or 10% Fetal Bovine Serum, on MDAEMB-231 Cell Proliferation: Previous work presented has demonstrated that genistein will inhibit MDA-231 cell growth with an IC50 of ~25 pM. Those experiments utilized standard passing media which contained 10% FBS plus genistein at 0, 10, 20, 40, or 80 pM. It was of interest to determine the effect of genistein upon cell growth in media containing a lower percent of FBS. FBS contains various growth factors that are required for the maintenance and growth of cells in culture and it also contains serum albumin. In the past I have demonstrated a FBS dependent increase in cell proliferation with increasing percent of FBS in the media: this suggests increased mitogenic stimulation of the tumor cells in the presence of increased growth factors. It was of interest to determine the effect of genistein upon cell proliferation in the presence of media containing different amounts of FBS. Will genistein inhibit cell proliferation at the same IC50 when the FBS concentration is lowered to 1%? 122 123 Experimental Protocol: 3-5-95 3-6-95 3-8-95 3-9-95 3-10-95 3-11-95 3-12-95 3-13-95 3-14-95 3-15-95 collected MDA-231 cells from 4 p100 plates, determined cell concentration and plated cells in 24—well plates at 15k cells per well in MEM containing 10% FBS changed media to MEM containing 0.1% FBS collected a plate to determine day 0 values by analyzing DNA content, began treatment of cells with MEM containing either 10% FBS or 1% FBS with 0, 10, 20, 40, or 80 pM genistein collected plates from the 10% and 1% FBS groups and ran DNA assays to assess cell proliferation, day 1 changed media in all plates collected plates from the 10% and 1% FBS groups and ran DNA assays to assess cell proliferation, day 3 changed media in all plates collected plates from the 10% and 1% FBS groups and ran DNA assays to assess cell proliferation, day 5 changed media in all plates collected plates from the 10% and 1% FBS groups and ran DNA assays to assess cell proliferation, day 7 124 Results: Genistein administered in MEM containing 10% FBS inhibited cell proliferation with an ICso of ~25 pM (Figure A1) which agrees with previous studies. In the MEM containing 1% FBS, genistein inhibited cell proliferation with an IC50 of ~10 pM (Figure A2). Conclusion: The results of this study suggest that the ability of genistein to inhibit cell proliferation is dependent upon the concentration of mitogens in the media. This is important when attempting to compare in_yitrg results to the in yiyg condition. The relationship between the concentration of growth factors in the media to the concentration found in 2129 is unknown. Furthermore the concentration in vivo is continually changing. The inhibitory affect of genistein seen in this study is dependent upon the concentration of mitogens the cells are exposed to, so although we see ICw's ranging from ca. 10 to 25 pM depending on culture conditions, the inhibitory concentration required or expected in yivg is not known. 125 6000 I? /’/" ///- —+— 0.1% FBS 4800 - // / / -<>- 10% ms , . / / , . g 3600 P d / W —a—- 10 pm GEN Q I/ :/ / ,’ / / a // 1' / _v- 20 UM GEN ‘3 2400 ~ [I / / / '/ / /'/ v -{}- Mauucnm / / / zoo - d/ V / //EJ _,_ 1 ’zé"'/ B// - 80 UN GEN /,/-’ f, /£;-"B” _____..—-? 1__¢-gggfik=——-u—f~——-~—~y—— 0 l 1 l 0 1 3 5 '7 Days Figure A1. Effect of genistein on MDA-MB-231 Cell ‘Proliferation. MDA-MB-231 human breast cancer cells were treated with 0, 10, 20, 40 and 80 pM genistein in MEM containing 10% FBS for a period of 7 days. Fluorometric analysis of cellular DNA was performed on days three and seven. Solid bars represent cells cultured for three days and open bars are data collected after seven days. Each point represents the mean i SEM of eight measurements. 126 4500 Bars I! SEM gD / / —+—- 0 1% 3500 ~ ,’ / / -<>— 1% / l/ g 2700 ~ / —a—- 10 uM can o [E a. //’ —V— 20 UM GEN ‘3 1800 ~ / / ./A —-B— 40 uM om / ./ / ,/ / ,/ 900 — [/0 .,-/-"" —+-- 80 tan or»: Figure A2. Effect of genistein on MDA-MB-231 Cell Proliferation. MDA-MB-231 human breast cancer cells were treated with 0, 10, 20, 40 and 80 pM genistein in MEM containing 1% FBS for a period of 7 days. Fluorometric analysis of cellular DNA was performed on days three and seven. Solid bars represent cells cultured for three days and open bars are data collected after seven days. Each point represents the mean i SEM of eight measurements. Appendix 8 Kinetic Analysis of B-Glucoronidase: Optimal ph for B-glucoronidase function is approximately pH 5. At this ph the isolation and preparation of serum samples for HPLC analysis of genistein produce chromatograms which are very ‘dirty’, however at approximately ph 7 the chromatograms look good. However, the activity of B—glucoronidase at this pH in serum samples was not known. Therefore the following study was conducted to assess B-glucoronidase activity at the higher ph. Experimental Protocol: Rat serum, from a study in which rats were gavaged with 40 mg genistein, was aliquotted into three 550 pl samples in 1.5 ml microfuge tubes. One tube was served as the control, no enzyme, one tube received 0.5 ul enzyme/10 ul serum (1x) and another tube received 1.0 ul enzyme/10 ul serum (2x). 50 ul samples were taken from all tubes at time 0, 15 and 30 min., and 1, 2, 4, 8, 12, 24 and 48 hours. Following the incubation, 50 pL of absolute methanol was added to each tube, the tubes vortexed and then centrifuged at 15,000 x g for 10 min. Approximately 75 pL was removed and placed at - 20%: until analysis. For analysis of genistein the 127 128 microfuge tubes were centrifuged at 15,000 x g for 10 min. and 20 pL injected onto a C18 column (Microsorb-MV, 5 pM 100A, Rainin Instrument in Woburn, MA) with a flow rate of 1.0 ml/min of 50:50 methanol:water with 0.1% acetic acid. Results: see Figure B. Conclusion: The data suggests the reaction had gone to completion in approximately 24 hours in the 2x reaction while it took around 48 hours in the 1x reaction. This study shows that the enzyme is functional at pH 7, the concentration required for greater effect, and the time required for completion of the reaction. These data will be used in future procedures for analyzing plasma genistein. 129 so 2x1numus 40— Ey—4} .......... “ /// [A"" o ,’ — I ,l ,5 3o —- ,’ ,’ g / ,’ rxnmmna: 5 ¢ A, 2 20 ~,’ ,’ I A, é / I I l’ 10 (A d’ tEerzna: 0 ,A——+~Lq 4————r— 1 1 . s_+ o 10 20 30 4o 50 Time in Hours Figure B. Kinetic analysis of B-glucuronidase in serum from rats gavaged with genistein. treated with B-glucuronidase Units) or without, Serum samples (50 ul) were (208 (1X) or 515 analyzed at for genistein with HPLC. (2X) Fishman incubated for different times and samples Appendix C Food Consumption Pattern in the Mbuse: Although feeding 750 pg/g genistein does not affect the growth of existing tumors feeding higher amounts of genistein may be effective. It is necessary to determine the plasma concentration of genistein in mice fed various amounts of genistein in order to evaluate the plausibility of feeding higher amounts...is there a plasma genistein concentration that could be effective. In order to determine the time where plasma genistein concentration reaches a maximum it is necessary to establish the food intake pattern of the mouse. Experimental Protocol: 18:00 provide 2 cups of food to mice, 5 per cage, 4 cages 20:00 weigh food cups 24:00 weigh food cups 06:00 weigh food cups 12:00 weigh food cups 18:00 weigh food cups 130 131 60 Five mice per cage so _ Total Food Disappearance 0 E E cage 1 1 ‘° ’ s; - cage 2 .2 261g G 30 r a 8 E: cage 3 (a. 14.8 g "5 (1mm ..... o 36.4 g I- 0 a. 10* ‘ _ I 18:00-20:00 20:00-24:00 24:00-06:00 06:00-12:00 12:00-18:00 Time Figure C. Food intake of mice over a 24 hour period. Two cups of food were weighed and provided to each cage of five mice at 6:00 pm. Food disappearance was monitored by weighing the food cups at the indicated times. At 6:00 am new cups were provided. 132 in: Q l 60 Five mice per cage 50 1 F Total Food 8 I lhmmmuwume g [:1 c... I- 27.3 {-5.40 L g g g- g a... 5 E 26.1 g tafllt g: ‘E E; illll cage ‘8 g 14.8 g E 2“ ” E W cage 9 E 36.4 g t- = o as n- e 1...- . lllll I 18:00-20:00 20:00-24:00 24:00-06:00 06:00—12:00 12:00-18:00 Time Figure C. Food intake of mice over a 24 hour period. Two cups of food were weighed and provided to each cage of five mice at 6:00 pm. Food disappearance was monitored by weighing the food cups at the indicated times. At 6:00 am new cups were provided. Appendix D Athymic Mice Fed Genistein for 10 Weeks: Genistein at 750 pg/g did not affect the growth of tumors in the above study. Feed intake was measured over three days and was similar among the groups. Weight change was also similar among the groups. To assess the effect of higher concentrations of genistein upon feed intake in the athymic nude mouse a feeding study was conducted. Experimental Protocol: 1-16-95 Receive 12 mice at 21 days of age and grouped 2 mice/cage, 2 cages/group. 1-31-95 Began feeding AIN-93G, AIN-93G + 1500 pg/g genistein and AIN-93G + 3000 pg/g genistein 2-1-95- Began assessing feed intake three days of every week for ten weeks 4-13-95 Killed and weighed all mice, weighed uterus and collected mammary gland for analysis of development Results: Food Intake: Food intake was 10.17 i .17 g/cage/day in the control group, 8.82 i .14 g/cage/day in the 133 134 1500 pg genistein/g group and 7.43 i .16 g/cage/day in the 3000 pg/g genistein group. (See Figure D) Weight Change: There were no significant differences in weight change among the groups, control i 4.11 g i .57, 1500 pg/g 5.26 g i .35 and 3000 pg/g i 4.24 i .76 (See Figure D). Uterus: Uterine weight in the treatment groups was not significantly different from the control: control 0.09 i .04, 1500 pg/g 0.10 i .02 and 3000 pg/g 0.86 i .01. Mammary Gland: The inguinal mammary gland was removed from each mouse and stained. Glands were analyzed and scores from 1 to 4 assigned for endbud analysis and ductal growth. Endbud Analysis: There were no significant differences among the treatment groups: control 1.75 i 1.5 (SD), 1500 pg/g 1.5 i .58 and 3000 pg/g 2.88 i .63. Duct Analysis: There was no significant difference in duct infiltration into the mammary fat pad among the control and the 1500 pg/g groups, control 3.95 i .06 and 1500 pg/g 3.63 i .48. The 3000 pg/g group did have a significant decrease in ductal 135 development compared to the control, control 3.95 i .06 and 3000 pg/g 2.88 i .63. 30 20 2 cages/treatment 2micelcage m 24 "- Mouse Weight 0 ..... 0 ____ 0,-“0 """ 0 16 g '0- Control Weight 5 .o ---- g'L'QA—“A‘r-Ax'A-‘fiu— a. 5 X",B:;__e--O———e-_€___e__€,-—<> ,_ -A- GEN Isooppm // 5 Weight ,5 1s ’0 a 12 g _o_ .= .‘2 ; . e . e a we“: on , e .4 e . .. O 9 e \ .5 & 1" ’:\ A. . f’K f‘ 9 "' Control 3 12 ‘ g 1 . rk‘ A! / _ 8 "3 Food Intake 8 fflx f.\.. _. ‘1.” GEN 1500mm 3 Food Intake ' ‘ . 3 Food Intake 2 0° '0' GENJOOOppm 6 " - 4 Food Intake o11111111111111111111111111 0 l 2 3 4 5 6 7 8 9 10 Weekof Measurement Figure D. Food intake and weight of mice fed 0, 1500 and 3000 pg genistein/g food. Athymic mice were fed genistein at the doses noted for a period of ten weeks. Food disappearance was monitored for 3 days of each week and weight assessed weekly. Open figures are mouse weight and closed figures are food intake per cage. 136 Conclusion: This study was conducted to determine food intake of mice fed 1500 and 3000 pg/g genistein over a long period (10 weeks). Although food intake was 2.7 g/cage/day (averaged throughout the experiment) (ps 0.001) less in the mice receiving 3000 pg genistein/g AIN-936, compared to control, and 1.35 g/cage/day in the mice receiving 1500 pg/g diet, weight change between the groups was not significantly different. However, the mice in the genistein treated groups weighed less at the beginning of the study than the mice in the control group: control 19.4 g, 1500 pg/g 17.6 g and 3000 pg/g 16.3 g. This could have affected weight change in the experiment. These data suggest food intake decreases with increasing concentration of genistein in the diet; however, the change in food intake does not produce an immediate change in weight gain. Appendix E Plamma.Analysis of Genistein frchMice Given a Bolus of 6 mg Genistein: The uptake, availability and metabolism of dietary genistein are unknown. This study sought to determine the point at which the maximum concentration of genistein would be present in the plasma from a bolus of 6 mg genistein in DMSO. This information will be used to determine the feasibility of this approach in giving genistein to mice in future tumor studies. Experimental protocol: 09:00 6 mg genistein administered as a bolus via gavage 10:00 kill three mice and collect plasma 11:00 kill three mice and collect plasma 13:00 kill three mice and collect plasma 17:00 kill three mice and collect plasma 21:00 kill three mice and collect plasma 09:00 kill three mice and collect plasma 137 138 Results: Genistein is absorbed into the blood within 1 hour after administration and reaches a peak at 4 hours (Figure E). Concentrations in excess of 20 pM are present for at least 12 hours and decrease to about 3 pM after 24 hours. Conclusion: This approach will produce plasma genistein concentrations in excess of 20 pM for at least 12 hours; however, it is not practical to gavage mice daily over the course of a long term study due to a potential mortality occurring during the gavage procedure. 139 60 Bars = SEM I n 8 3 mice 20* Plasma [Genistein] in uM 8 l 10' 0 1 1 1 1 1 1 1 1 1 1 1 0 2 4 6 8 10 12 14 16 18 20 22 24 Time Figure E. Plasma genistein in mice. Balb/C mice were 'gavaged with 6 mg genistein in 100 ul DMSO and killed at the indicated time points. Blood was collected and analyzed for genistein with HPLC. Each point represents the mean plasma genistein concentration of 3 mice. Appendix F Gavage vehicle and Its Affect on Food Intake in the Mouse: Earlier experiments in which genistein was gavaged to rats and mice utilized DMSO as a vehicle for the genistein. Given the effect of caloric restriction on tumor growth it was necessary to assess the effect of DMSO on food intake. The following experiment was conducted to evaluate the effect of DMSO on food intake. Experimental Protocol: Two cages each containing five mice were included in the study. Mice in one cage were gavaged daily at 14:30 with 100 pl DMSO. Mice were then placed in new cages with new food cups, two per cage, and the food cups weighed. Each day for 3 days mice in the one cage were gavaged. Each 24 hour period the cups were removed from both cages and weighed. After 3 days the gavaging was discontinued but food intake monitored for an additional 5 days. Three days later the mice initially receiving no gavage were gavaged with 100 pl corn oil for two days. 140 141 Results: CAGE TREATMENT MEAN FOOD INTAKE/CAGE (SEM) 1 DMSO Gavage (3 days) 12.6 (2.22) 1 no gavage (3 days) 23.9 (1.4) 2 no gavage (5 days) 24.5 (1.38) 2 corn oil gavage (2 days) 19.7 (3.65) Food disappearance see Figure F Conclusion: The data indicate that gavaging reduces food intake especially with the DMSO gavage. If gavaging is to employed in future experiments to administer genistein the vehicle used is important. DMSO resulted in a large decrease in food intake whereas the corn oil slightly decreased food intake. Genistein is not soluble in corn oil and the suspension is too viscous for use in the gavage tube so an alternate vehicle had to be found. Medium chain triglyceride oil was found to be marginally useful. Genistein could be added to MCT—oil at 6 mg/100 pl with the resultant suspension capable of passing through the gavage tube however with difficulty at times. 142 30 Bars=SEM E 5micelcage _ 23 o __ 4L .5 an 20 _ _ DMSO a ” sfif % we" 1:] DMSO CONTROL 0 1 = 1: E CORN OIL a 0 g 10 _ E: CORN OIL CONTROL a .2 r :1 “O O O c. 0 w . Cage 1 Cage 2 Figure F. Food intake in mice gavaged with corn oil or DMSO. Food intake was monitored in five mice (cage 1) that .were gavaged with 100 pl DMSO for 3 days and then monitored for an additional 3 days while not gavaged. Food intake was monitored in another group of 5 mice (cage 2) for 5 days and then the mice were gavaged with 100 pl corn oil for a 2 day period and food intake monitored. Data are expressed as mean food intake per group. ' Appendix G Genistein Gavaged to Tumor Bearing Mice: A previous study had found that plasma genistein concentrations in excess of 20 pM could be achieved in mice by gavaging 6 mg genistein. This study was conducted to assess the feasibility of this approach over a long period and also to assess the effect upon tumor growth. Experimental Protocol: 3-6-96 3-9-96 4-22-96 4-24-96 4-29-96 Results: Mice were received at 23 days of age. Tumor cells injected at 26 days of age. 100% take rate. Began gavaging mice daily with 6 mg genistein in 100 pl MCT-oil or 100 pl MCT—oil, 2 cages 5 mice/cage, measured food disappearance daily Killed mice, measured tumors, collected tumors and blood. Change in tumor area see Figure Gl Plasma genistein concentration see Figure GZ Food intake see Figure G3 143 144 50 4 mice/cage Bars = SEM 30* 10* MEAN TUMOR AREA CHANGE IN mm2 6 mg Genistein Control Figure Gl. 'Tumor area change. Tumor bearing athymic mice (4 mice/group) were gavaged with 6 mg genistein in 100 pl MCT-oil or 100 ul MCT—oil for a period of 6 days. Data is expressed as mean tumor area change per group. 145 12 n=4mke _— Bars = SEM 10 - Plasma Genistein in uM ON I oj Free genistein Total genistein Figure GZ. Plasma genistein in mice. Tumor bearing athymic mice (4 mice/group) were gavaged with 6 mg genistein in 100 pl MCT-oil or 100 p1 MCT-oil for a period of 6 days. Blood was collected and analyzed for genistein with HPLC. Each point represents the mean plasma genistein concentration of 3 mice. 146 40 Bars = SEM 4 mice/cage g 30 t 5 - 6 mg Gen-MCT-Oil .5 l cage 0 a: 20 _ D MCT-Oil E) 1 . l cage ‘3 W [MW Control E 10 __ 3 cages Ln 0 1 —1 Figure G3. Food intake. Tumor bearing athymic mice (4 mice/group) were gavaged with 6 mg genistein in 100 pl MCT-oil or 100 pl MCT-oil alone for a period of 6 days. Food intake was determined for 4 days in a control group in addition to the two gavage groups. Data are expressed as mean food intake per cage over 4 days. Asterisk denotes significance at ps.05. 147 Conclusion: These results suggest that this dose of genistein could have an effect upon tumor growth although the difference in this study was not significant. Even though the control mice consumed significantly (ps .005) less food tumor growth appears to be more rapid in this group. Gavaging 6 mg genistein in MCT-oil produced plasma genistein concentrations of ca. 10 pM, 4 hours after gavaging, a value much less than that seen in the DMSO gavage study (ca. 47 pM). It is possible that DMSO might facilitate the absorption of genistein resulting in higher plasma concentrations. Gavaging is not an appropriate means of administering chemicals over a long period of time for the following reasons: 1) damage to the mice, 2) potential fatality to mice due to the procedure, and 3) stress on the mice. In addition the resulting genistein/MCT-oil suspension was at times difficult to pass through the gavage tube. Higher concentrations of genistein in MCT-oil are unlikely to be successful. Appendix H Genistein Release from Elvax Pallets: To assess the release of genistein from elvax pellets the following study was conducted. Experimental Protocol: Genistein containing elvax pellets were incubated in phosphate buffered saline and samples taken periodically. Samples were then dried, brought up in methanol, and analyzed by high pressure liquid chromatography (HPLC). 40 ul were injected onto a C18 column (Microsorb-MV, 5 uM 100A, Rainin Instrument in Woburn, MA) with a flow rate of 1.0 ml/min of 50:50 methanol:water with 0.1% acetic acid. Results: Genistein is released from the pellet at the following rate: ELAEEBD_IIME EEBQENI_QE_BEMAIHIN§_§EN_BELEA§ED 4 hrs 26 24 hrs 38 48 hrs 33 168 hrs 22 Total release after 168 hrs is equivalent to 75% of the genistein content of the pellet. See Figure H. 148 149 Conclusion: Genistein is released from the plastic elvax matrix into an aqueous environment. U 5 i I Remaining Genistein in ug N I t 0 l l l l 11 l 7, 0 12 24 36 48 I60 164 I68 Time in Hours Figure B. Genistein release from an Elvax pellet. A 1.9 mg Elvax pellet containing 3.77 pg genistein was incubated in phosphate buffered saline at 370C and samples withdrawn at the indicated times and analyzed for genistein with HPLC. Appendix I Effect of Genistein Pellet Implants on Mammary Gland Development in the Mouse: The following study was conducted to assess the effect of genistein (GEN) on the mammary gland of female intact and ovariectomized mice. Dr. Haslam's laboratory personnel collaborated in the experiment. Intact and ovariectomized (OVEX) immature female mice were implanted with elvax pellets containing either genistein (5 ug) or estradiol (E2)(5 ng) into the number four mammary gland fat pad. The contralateral mammary gland was implanted with an elvax pellet and served as the control. Experimental Protocol: 2-15 ovariectomized group DOB 3-2 intact group DOB 3-23 ovariectomies performed 4—3 pellets prepared 4—7 mice were divided into the following groups and the following treatments initiated group 1 (OVEX) implanted with elvax pellets containing 5 ug GEN 150 151 group 2 (OVEX) implanted with elvax pellets containing 5 ng E2 group 3 intact implanted with elvax pellets containing 5 ug GEN group 4 intact implanted with elvax pellets containing 5 ng E2 4-11 mice were killed and mammary tissue mounts prepared Results: There were 5 mice per group. The mammary gland mounts were examined for end bud development and number, and ductal growth. Each mouse was implanted with the plastic matrix alone into the other number four mammary gland fat pad and thus served as its own control. group 1: (OVEX) Genistein had no effect compared to the control gland (Figure I1) group 2: (OVEX) E2 stimulated development compared to the control gland (Figure 11) group 3: (Intact) Genistein did not stimulate development compared to the control gland and appears to have slightly inhibited development (Figure 12) group 4: (Intact) E2 did not stimulate development compared to the control gland (Figure 12) 152 3 - Elldblld Bar: 3 SEM 2 [:1 D... —— __ O 0 CD E __ o 2 r g- __ G. l .2 0 _h—— > I 0 G: E‘ a 1” E E a E 0' .. . . 5 ng Estrogen Estrogen Control 5 ng Genistein Genistein Control Treatment Figure I1. Effect of genistein pellet implant on the mammary gland in ovariectomized mice. Female Balb/C mice (n=5) were ovariectomized at 36 days of age. Plastic pellets containing either 5 ng estradiol or 5 ug of genistein were implanted into the mammary gland, at 51 days of age, for a period of 5 days. The contralateral mammary gland was implanted with plastic pellets to serve as a control. The mammary glands were removed, stained and analyzed for development by assessing endbud size and number and extent of ductal growth. 153 - Endbud Bars 8 SEM [:3 Duct Mammary Development Score '0 I 5 ng Estrogen Estrogen Control 5 ug Genistein Genistein Control Treatment Figure 12. Effect of genistein pellet implant on the mammary gland in intact mice. Plastic pellets containing either 5 ng estradiol or 5 ug of genistein were implanted into the mammary gland of 36 day old female Balb/C mice (n=5) for a period of 5 days. The contralateral mammary gland was implanted with plastic pellets to serve as a control. The mammary glands were removed, stained and analyzed for development by assessing endbud size and number and extent of ductal growth. 154 Conclusion: Genistein containing plastic pellets, implanted into the mammary gland of the mouse, does not appear to stimulate development of the mammary gland in ovariectomized or intact mice. Genistein appears to have slightly inhibited development in the intact mouse. These results suggest genistein is potentially capable of inhibiting the growth of mammary tissue in 1119. Appendix J Genistein/Cholesterol Pellet Implant into Tumor bearing Mice: Dietary genistein (750 ug genistein/g diet) does not appear to affect the growth of existing tumors and administration of genistein by gavage is not practical; therefore, genistein pellets were made and implanted into the mice at the site of the tumor. Composition of the pellets was 10 mg genistein and 20 mg cholesterol or 30 mg cholesterol. The cholesterol matrix was chosen over elvax because the pellets would have been too large with the elvax. Experimental Protocol: 3-19-96 received 4 mice at 28 days of age 3-26-96 injected tumor cells 5-16-96 implanted genistein pellets in two mice and cholesterol pellets in 2 mice between the body wall and the tumor at each tumor site 5-31—96 collected blood, measured and collected tumors 155 Results: 1. The percent genistein In addition presented below. beginning 156 of the study. increase in tumor area was 64% for the pellet group and 60% for the control. pellet weights were assessed and are All pellets weighed 30 mg at the Genistein was not detected in the plasma of the mice implanted with pellets containing genistein. Genistein pellets 1) 30.4 2) 27.2 (pellet 3) 28.8 4) 30.5 (pellet 5) 26.1 (pellet 6) 31.5 (pellet 7 and 8 (pellet part Conclusion: chipped) discolored) chipped) discolored) chipped, missing) Cholesterol pellets 29. 30. 29. 30. 30. 28. 25. 26. 1 2 U" U'l (pellet discolored) (pellet discolored) (pellet discolored) (pellet chipped, discolored) (pellet discolored) The experimental groups consisted of only two mice. The study was conducted to assess the viability of this approach. There were two tumors on two mice that were necrotic so these mice were separated into the two treatment 157 groups. As a result beginning tumor area was not equal among the groups. The group receiving the genistein pellets had a lower mean tumor area, 65.89 nmfi compared to 106.2 mm2 in the control. Pellet weights changed very little suggesting little release of genistein from the pellet. However it is difficult to gain much information from pellet weights due to a couple of factors. Chipping of the pellet during implantation or removal results in loss of the pellet which could lower the weight. Also while in the mouse the pellet could possibly increase in weight through infiltration of substances from the body as well as encapsulation material on the outside of the pellet which is difficult to remove. The implantation process itself damages the host tumor environment which in itself could affect the growth of tumor. This is a major limitation in employing this approach to study changes in tumor growth with respect to a control. Appendix K Genistein Pellet Implanted into Mice: The previous pellet implantation study did not show an effect on tumor growth. The lack of an effect could have been due to genistein not releasing from the cholesterol matrix therefore the following study was conducted. Pure genistein pellets were made and implanted subcutaneously on the backs of athymic mice. Pellets remained in the mice for a period of 17 days after which time they were removed and weighed. Results: INITIAL/FINAL MOUSE WEIGHT INITIAL/FINAL PELLET WEIGHT 20.2 / 21.6 g 23.8 / 24.5 mg 18.6 / 20.4 g 24.1 / 22.2 mg 18.6 / 20.5 g 37.2 / 36.1 mg 20.6 / 22.5 g 37.7 / 36.1 mg (pieces) Conclusion: Pellet weight did not change appreciably over the 17 days they were implanted in the mice. Furthermore some of the pellets actually weighed more. The pellets remained intact 158 159 for the most part with the exception of some chipping during the implantation and removal procedure. It appears that genistein is not released from the pellets. This could be due to the compact nature of the pellets. In order to get genistein to remain in pellet form a large amount of force is required to compress the genistein. List of References 160 161 List of References Adams, N.R. (1989). Phytoestrogens. In: Toxicants of Plant Origin (Cheeke, P.R., ed.), Vol. IV, Chap. 2: 23-51. 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