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Cancer Res. 2001 Jul 1;61(13):5045-50. We have demonstrated that the isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo (C. Y. Hsieh et al., Cancer Res., 58: 3833-3838, 1998). The isoflavones are a group of phytoestrogens that are present in high concentrations in soy. Whether consumption of genistein from soy protein will have similar effects on estrogen-dependent tumor growth as pure genistein has not been investigated in the athymic mouse tumor implant model. Depending on processing, soy protein isolates vary widely in concentrations of genistein. We hypothesize that soy isolates containing different concentrations of genistein will stimulate the growth of estrogen-dependent cells in vivo in a dose-dependent manner. To test this hypothesis we conducted experiments in which these soy protein isolates were fed to athymic mice implanted s.c. with estrogen-dependent tumors. Genistein content (aglycone equivalent) of the soy isolate diets were 15, 150, or 300 ppm. Positive (with 17beta-estradiol pellet implant) and negative (no 17beta-estradiol) control groups received casein-based (isoflavone-free) diets. Tumor size was measured weekly. At completion of the study animals were killed and tumors collected for evaluation of cellular proliferation and estrogen-dependent gene expression. Incorporation of bromodeoxyuridine into cellular DNA was used as an indicator of cell proliferation, and pS2 mRNA was used as an estrogen-responsive gene. Soy protein diets containing varying amounts of genistein increased estrogen-dependent tumor growth in a dose-dependent manner. Cell proliferation was greatest in tumors of animals given estrogen or dietary genistein (150 and 300 ppm). Expression of pS2 was increased in tumors from animals consuming dietary genistein (150 and 300 ppm). Here we present new information that soy protein isolates containing increasing concentrations of genistein stimulate the growth of estrogen-dependent breast cancer cells in vivo in a dose-dependent manner. From the full text article: The Food and Drug Administration recently approved a health claim for soy protein. The claim states "25 g of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease" (1). As a result, the consumption of soy protein by Americans has increased. Soy and soy-derived supplements are not being used solely to lower serum cholesterol and reduce risk of heart disease. Soy contains a complex mixture of a variety of phytochemicals; our focus is on the estrogenic isoflavones referred to as phytoestrogens. Isoflavones have been demonstrated to act as estrogen agonists by binding to the estrogen receptor and generating estrogen-induced responses (2, 3, 4). As a result, postmenopausal women may consume soy for the estrogenic effects of these compounds to relieve menopausal symptoms. Soy products are marketed as a "natural" alternative to hormone replacement therapy with the perception that these phytoestrogens are without the risks associated with hormone replacement therapy. Isoflavone-containing products are produced in various forms, including supplement capsules, which contain mixtures of isoflavones derived from soy. Isoflavones are also present in bioactive concentrations in food products such as soy protein isolates, which can contain varying isoflavone content. It is generally accepted that consuming the phytochemical components of soy, particularly the isoflavones, in pure form, as in supplements, may pose some health concerns but that consumption of more whole foods containing these are natural and, as such, safe. It is important to note that the concentration of these compounds in soy protein isolates is dependent on the method of processing, and specific processing methods can be used to enrich the isoflavone content in soy protein isolates. Clinical and preclinical laboratory animal and in vitro studies have demonstrated the hormonal activity of dietary isoflavones. Our laboratory has examined the estrogenic activity of genistein. In vitro, when human estrogen-dependent breast cancer (MCF-7) cells were treated with increasing concentrations of genistein (1–10 µM), the cells expressed increased mRNA levels of pS2, an estrogen-responsive gene, in a dose-dependent manner. In vivo, 25-day-old ovariectomized, athymic mice consuming dietary genistein for 5 days had increased number and size of terminal end buds in the mammary gland when compared with control animals(5) . Not only do the isoflavones have estrogenic activity on various tissues when given in pure form but also when consumed in products such as soy protein isolate. In humans, Petrakis et al.(6) demonstrated that consumption of soy protein isolate had stimulatory effects on the breast tissue of premenopausal women. They found that duct fluid aspirates contained greater numbers of hyperplastic epithelial cells in women consuming soy protein isolate. Nipple aspirate apolipoprotein D and pS2 expression has also been shown to be elevated in women consuming 60 g of soy in the form of ground, textured vegetable protein for as little as 2 weeks(7) . These data collected from both laboratory animals and humans demonstrate the estrogenic activity of the isoflavones whether they are taken as a pure compound or consumed in soy protein isolate containing isoflavones. The role that isoflavones play in breast cancer is unclear. Some reports indicate that exposure to genistein is preventative in the development of breast cancer (8, 9, 10), whereas others show that genistein stimulates the growth of existing estrogen-dependent tumors (5). Studies published over the last 5 years have demonstrated that exposure to dietary genistein before puberty reduces the number of chemically induced mammary tumors formed in female Sprague Dawley rats. The authors suggest protection against the development of breast cancer tumors is attributable to the estrogenic effects of genistein that causes increased cellular differentiation in mammary gland cells of prepubertal animals. A differentiated cell undergoes less proliferation and therefore is less likely to progress through the cancer process (8, 9, 10). If prepubertal exposure to genistein results in earlier differentiation of the mammary gland, then the assumption can be made that earlier differentiation would be protective against chemically induced mammary tumors. Cohen et al. (11) found that continual feeding of soy protein isolate (containing 1.67 mg total isoflavones per g isolate) postpubertally for 18 weeks after chemical induction of mammary tumors in rats had no detectable effect on the development of these tumors. Our research has focused on the effect dietary genistein has on the growth of existing estrogen-dependent (MCF-7) tumors in animals with low circulating E24 concentrations. We have demonstrated that genistein fed at 750 ppm stimulates the growth of MCF-7 cells implanted into athymic mice. Mice consuming 750 ppm dose of genistein have a total plasma genistein concentration of 2 µM (5). This is a relevant dietary dosage because women who consume varying amounts of isoflavones from soy milk have plasma genistein levels of 0.8-2.2 µM (12) . In vitro genistein has been shown to stimulate the growth of MCF-7 cells at concentrations as low as 200 nM (2, 5). Whereas these data demonstrate genistein can stimulate the growth of estrogen-dependent breast cancer tumors, it has not yet been determined if soy protein isolate, which contains a complex mixture of phytochemicals, can exhibit similar effects on growth of estrogen-dependent tumors. [...] By week 3 after retreatment with a new 2-mg E2 pellet, the average cross sectional area of the tumors in the positive control group was 126 mm2 (Fig. 1, A and B). At this point, these mice were killed. By week 12 after the E2 pellets were removed, the negative control tumors regressed to an average area of 12 mm2. They then maintained this size until termination of the study. Soy protein isolates stimulated growth of MCF-7 tumors in a dose-dependent fashion (Fig. 1A). Also, casein-based diets containing equivalent levels of genistein stimulated tumor growth in a dose-dependent manner (Fig. 1B). By week 29 after E2 pellet removal, both the LSI and LG groups were similar to the negative control group resulting in average tumor areas of 16 mm2 and 14 mm2, respectively. The average tumor area in the MSI and MG groups were significantly higher than the negative control group with final cross sectional areas of 60 mm2 and 54 mm2 (P < 0.01) respectively. The MSI and MG groups were not significantly different from one another. The tumors from both the HSI and HG groups were significantly larger than the negative control and other dietary treatment groups (P < 0.01). The HSI average area was 112 mm2 whereas the HG group had a final average area of 97 mm2. These (HSI and HG) were not significantly different from one another and were similar to that obtained from the positive control group 3 weeks after retreating them with E2 pellets (Fig. 2). Body weight was monitored weekly, and no significant difference was observed among the treated and control groups (data not shown). These data indicate that dietary soy isolates containing increasing concentrations of genistein and casein-based diets containing equalized concentrations of genistein act in a dose-dependent manner to stimulate growth of human estrogen-dependent breast cancer cells transplanted into athymic mice. Also, it is important to note that there were no significant differences in growth of tumors in the negative control, low soy isolate, and low genistein groups. These data suggest that there is a threshold level of dietary genistein below which no increase in estrogen-dependent tumor growth is observed. [...] The purpose of this study was to determine the influence of dietary soy protein isolates containing increasing concentrations of genistein on the growth of estrogen-dependent human breast cancer cells transplanted into athymic mice. The results presented here demonstrate that soy protein isolates containing varying concentrations of genistein can stimulate growth of estrogen-dependent tumors similar to that seen with pure dietary genistein. Soy protein isolates stimulated the growth of MCF-7 tumors in a dose-dependent manner as the concentration of genistein increased in the isolates. Tumor growth was significantly increased in animals consuming genistein at concentrations of 150 and 300 ppm in both the casein- and soy-based diets. Dietary concentrations in this study are lower than previous studies in which MCF-7 tumor growth was observed in athymic mice consuming dietary genistein at 750 ppm (5). In the study presented here, tumors took longer to reach a maximum size when compared with the previous study, which was likely attributable to the lower concentrations of genistein in the diet. Cellular proliferation was increased in a dose-dependent manner as genistein concentrations increased in both the soy protein and casein-based diets consistent with the tumor growth data. The increase in MCF-7 cell proliferation was likely attributable to an estrogenic effect as indicated by the modest increase in pS2 expression. These findings suggest that genistein, when fed to athymic mice in pure form or in a food source rich in the isoflavone such as soy protein isolate, can stimulate estrogen-dependent tumor growth in a dose-dependent manner. The estrogenic activity of isoflavones has been well documented in in vitro, in vivo, and clinical studies. Genistein can bind to the estrogen receptor with an affinity 100-1000 times lower than E2 (21). Additionally, in ovariectomized Sprague Dawley rats, dietary genistein (750 ppm) enhanced lobular-alveolar mammary gland development, increased uterine weight, and at 750 ppm, increased pituitary prolactin secretion and serum prolactin levels. In the uterus, dietary genistein increased c-fos mRNA expression (21). In vitro studies showing an increase in the levels of the estrogen-responsive genes pS2 and c-fos when cells are treated with genistein, provide additional evidence of the estrogenic property of genistein (5, 22). Clinical studies have also confirmed the preclinical studies evaluating the estrogenicity of genistein. Premenopausal women consuming textured vegetable protein containing 45 mg of isoflavones had prolonged menstrual cycles. Additionally, the follicular phase was extended by suppression of the normal surge of follicle-stimulating hormone and luteinizing hormone (23, 24). These data in conjunction with the observations that dietary soy increases cell proliferation in human breast tissue (6) and increases pS2 expression (7) demonstrates biological (estrogenic) activity in humans. Isoflavones are being marketed to postmenopausal women in both supplement form and in soy food products for the relief of the symptoms of menopause. It is, therefore, critical to understand what activities genistein may have in these women. Consideration must be given to how genistein may influence the endogenous levels of serum E2 in women consuming the compound. There is evidence suggesting diets high in soy have the ability to lower serum E2 concentrations (25 , 26). A study supporting this finding showed that normally cycling women consuming a soy diet containing 154 mg total isoflavones/day had a 25% reduction in circulating E2 (27). Contrarily, other studies in which premenopausal women have consumed soy have found no change in serum E2 concentrations (6, 28), and one study showed increased serum E2 in women consuming soy (23). It is unclear how isoflavones in soy effect normal circulating E2 concentrations in premenopausal women. Postmenopausal women consuming a soy diet have been shown to have no change in serum circulating E2 levels (28). This finding in conjunction with reports that genistein acts estrogenically, and postmenopausal women naturally have low plasma concentrations of E2, suggest that in a postmenopausal woman, these weak estrogens may have significant estrogenic activity. At concentrations above 10 µM genistein has been demonstrated to inhibit in vitro cell proliferation in a variety of cell types including estrogen-dependent (MCF-7) and estrogen-independent (MDA-468) human breast cancer cells (29). Genistein has also been shown to inhibit activities of protein tyrosine kinase at concentrations >20 µM (30). However, at levels as low as 200 nM, genistein stimulates the growth of MCF-7 cells in vitro (2, 5) and in vivo at 1 µM (5). In MCF-7 cells, at concentrations from 20–90 µM genistein inhibited DNA synthesis, but stimulated DNA synthesis at concentrations of 0.1–10 µM (31). In this study we demonstrated that dietary genistein consumed at 15 ppm, whether from soy protein or as a pure compound, does not stimulate growth that is significantly different from the negative control group lacking any form of estrogen exposure in regard to stimulation of MCF-7 tumor growth. These results suggest that there is a level of dietary genistein that will not lead to an increase in tumor growth. Additional investigation will be necessary to determine the concentration of this threshold dose. [...] Categories: 2001, Soy, Phytoestrogens, Cancer, Endocrine, Estrogen, DNA synthesis, Nutrition and diet |