Effects of Genistein on the Mammary Gland Proliferation of Adult Ovariectomised Wistar Rats

Abstract

The effects of phytoestrogens on the female breast are discussed controversially. On the one hand, epidemiological and experimental data provide evidence that dietary phytoestrogens may prevent the development of breast cancer. On the other hand, in breast cancer cell lines and tumour models isoflavone phytoestrogens have been demonstrated to stimulate the growth of estrogen-dependent breast cancer cells. To further investigate the molecular effects of genistein (Gen) on the mammary gland, we treated non-tumour bearing, ovariectomised female Wistar rats with this phytoestrogen either subcutaneously (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E₂, 0.004 mg/kg s. c.) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with Gen per os or s. c. results in a small but significant stimulation of the uterine wet weight. In the mammary gland, Gen stimulates the expression of progesterone receptor (PR) but, in contrast to E₂, the isoflavone does not stimulate the expression of PCNA. These findings resemble recent data demonstrating a differential ability of Gen to induce uterine gene expression and uterine proliferation. Our data indicate that in non-malignant breast tissue short-term administration of Gen, in contrast to more potent estrogens like E₂, does not induce proliferation. Chronic stimulation of proliferation is believed to be a key mechanism during the development of breast cancer. The limited ability of Gen to stimulate proliferation in this tissue could be an indication for a limited carcinogenic potency of Gen in the breast. In further investigations it is important to identify molecular differences between healthy and malignant breast tissue which may explain the different sensitivity towards Gen treatment.

Abbreviations

Gen:genistein

PCNA:proliferating cell nuclear antigen

E₂:17β-estradiol

ER:estrogen receptor

EE:ethynylestradiol

PR:progesterone receptor

HRT:hormone replacement therapy

OVX:ovariectomised

Key words

Genistein - breast cancer - phytoestrogens - proliferation - estrogens - mammary gland

References

• 1 Davis S R, Dalais F S, Simpson E R, Murkies A L. Phytoestrogens in health and disease.  Recent Prog Horm Res. 1999;  54 185-210, discussion: 210 - 1
• 2 Committee on Toxicity of Chemicals on Food/Consumer Products and the E nvironment. COT Report-Phytoestrogens and Health. London; Food Standards Agency 2003 http://www.food.gov.uk/multimedia/webpage/phytoreportworddocs
• 3 Hu S A. Risks and benefits of soy isoflavones for breast cancer survivors.  Oncol Nurs Forum. 2004;  31 249-63
• 4 Russell L, Hicks G S, Low A K, Shepherd J M, Brown C A. Phytoestrogens: a viable option?.  Am J Med Sci. 2002;  324 185-8
• 5 Squadrito F, Altavilla D, Crisafulli A, Saitta A, Cucinotta D, Morabito N. et al . Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study.  Am J Med. 2003;  114 470-6
• 6 Fanti P, Monier-Faugère M C, Geng Z, Schmidt J, Morris P E, Cohen D. et al . The phytoestrogen genistein reduces bone loss in short-term ovariectomized rats.  Osteoporos Int. 1998;  8 274-81
• 7 Heim M, Frank O, Kampmann G, Sochocky N, Pennimpede T, Fuchs P. et al . The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells.  Endocrinology. 2004;  145 848-59
• 8 Adlercreutz H. Phytoestrogens and breast cancer.  J Steroid Biochem Mol Biol. 2002;  83 113-8
• 9 Wu A H, Wan P, Hankin J, Tseng C C, Yu M C, Pike M C. Adolescent and adult soy intake and risk of breast cancer in Asian-Americans.  Carcinogenesis. 2002;  23 1491-6
• 10 Yamamoto S, Sobue T, Kobayashi M, Sasaki S, Tsugane S. Soy, isoflavones, and breast cancer risk in Japan.  J Natl Cancer Inst. 2003;  95 906-13
• 11 Bouker K B, Hilakavi-Clarke L. Genistein: does it prevent or promote breast cancer?.  Environ Health Perspect. 2000;  108 701-8
• 12 Lamartinière C A, Murrill W B, Manzolillo P A, Zhang J X, Barnes S, Zhang X. et al . Genistein alters the ontogeny of mammary gland development and protects against chemically-induced mammary cancer in rats.  Proc Soc Exp Biol Med. 1998;  217 358-64
• 13 Lamartinière C A. Protection against breast cancer with genistein: a component of soy.  Am J Clin Nutr. 2000;  71 1705-7, discussion: 1708 - 9
• 14 Cotroneo M S, Wang J, Fritz W A, Eltoum I E, Lamartinière C A. Genistein action in the prepubertal mammary gland in a chemoprevention model.  Carcinogenesis. 2002;  23 467-74
• 15 Ju Y H, Allred C D, Allred K F, Karko K L, Doerge D R, Helferich W G. Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice.  J Nutr. 2001;  131 2957-62
• 16 Allred C D, Allred K F, Ju Y H, Virant S M, Helferich W G. Soy diets containing varying amounts of genistein stimulate growth of estrogen-dependent (MCF-7) tumors in a dose-dependent manner.  Cancer Res. 2001;  61 5045-50
• 17 Allred C D, Allred K F, Ju Y H, Clausen L M, Doerge D R, Schantz S L. et al . Dietary genistein results in larger MNU-induced, estrogen-dependent mammary tumors following ovariectomy of Sprague-Dawley rats.  Carcinogenesis. 2004;  25 211-8
• 18 Allred C D, Allred K F, Ju Y H, Goeppinger T S, Doerge D R, Helferich W G. Soy processing influences growth of estrogen-dependent breast cancer tumors.  Carcinogenesis. 2004;  25 1649-57
• 19 Hilakivi-Clarke L, Cho E, Cabanes A, DeAssis S, Olivo S, Helferich W. et al . Dietary modulation of pregnancy estrogen levels and breast cancer risk among female rat offspring.  Clin Cancer Res. 2002;  8 3601-10
• 20 Winer J, Jung C K, Shackel I, Williams P M. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro .  Anal Biochem. 1999;  270 41-9
• 21 Diel P, Geis R B, Caldarelli A, Schmidt S, Leschowsky U L, Voss A. et al . The differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and proliferation in the rat is associated with a substance specific modulation of uterine gene expression.  Mol Cell Endocrinol. 2004;  221 21-32
• 22 Degen G H, Janning P, Wittsiepe J, Upmeier A, Bolt H M. Integration of mechanistic data in the toxicological evaluation of endocrine modulators.  Toxicol Lett. 2002;  127 225-37
• 23 Geis R B, Diel P, Degen G H, Vollmer G. Effects of genistein on the expression of hepatic genes in two rat strains (Sprague-Dawley and Wistar).  Toxicol Lett. 2005;  157 21-9
• 24 Diel P. Tissue-specific estrogenic response and molecular mechanisms.  Toxicol Lett. 2002;  127 217-24
• 25 Iatropoulos M J, Williams G M. Proliferation markers.  Exp Toxicol Pathol. 1996;  48 175-81
• 26 Degen G H, Janning P, Diel P, Bolt H M. Estrogenic isoflavones in rodent diets.  Toxicol Lett. 2002;  128 145-57
• 27 Ashby J. Testing for endocrine disruption post-EDSTAC: extrapolation of low dose rodent effects to humans.  Toxicol Lett. 2001;  120 233-42
• 28 Henderson B E, Ross R K, Pike M C, Casagrande J T. Endogenous hormones as a major factor in human cancer.  Cancer Res. 1982;  42 3232-9
• 29 Lupulescu A. Estrogen use and cancer incidence: a review.  Cancer Invest. 1995;  13 287-95
• 30 Liu B, Edgerton S, Yang X, Kim A, Ordonez-Ercan D, Mason T. et al . Low-dose dietary phytoestrogen abrogates tamoxifen-associated mammary tumor prevention.  Cancer Res. 2005;  65 879-86