The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Abstract for TestSmart -- Endocrine Disruptors

Aromatase as a Molecular Target of Natural and Synthetic Molecules

Robert W. Brueggemeier, Ph.D.
College of Pharmacy and OSU Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio

Estrogens are important female sex steroids that are necessary for female reproduction, and estradiol is the most potent endogenous estrogen produced in the body. Estradiol is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase, with the highest levels of enzyme present in the ovaries of premenopausal women, in the placenta of pregnant women, and in the peripheral adipose tissues of postmenopausal women and of men (1). Aromatase activity has also been demonstrated in breast tissue in vitro (2-8); furthermore, expression of aromatase is highest in near breast tumor sites. Certain human breast cancer cell lines also exhibit aromatase activity, and these cells are able to convert physiological levels of androgens into estrogens and elicit estrogen-mediated effects (9-11). The aromatase enzyme complex is bound in the endoplasmic reticulum of the cell and is comprised of two major proteins. One protein is cytochrome P450arom, a hemoprotein that converts C19 steroids (androgens) into C18 steroids (estrogens) containing a phenolic A ring. The second protein is NADPH-cytochrome P450 reductase, which transfers reducing equivalents to P450arom .

The gene expressing cytochrome P450arom is referred to as the CYP19 gene and is part of the cytochrome P450 superfamily. The cDNA for aromatase encodes a 55kDa protein containing 503 amino acids. The aromatase gene sequence exhibits high homology amongst various species, such as human, rat and mouse. The enzyme in humans is expressed in numerous tissues such as placenta, adipose, ovarian granulosa cells, testicular Sertoli cells, testicular Leydig cells, liver, and the brain. Since estrogen serves numerous roles in the various tissues, aromatase biosynthesis is very tissue-specific and tightly regulated (1,8,12).

Inhibition of aromatase or alterations in its level of expression in tissues can dramatically lower estrogen levels and affect reproductive capabilities. One desired therapeutic outcome from aromatase inhibition is the treatment of estrogen-dependent diseases, and several potent steroidal and nonsteroidal inhibitors of aromatase have been developed for estrogen-dependent breast cancer (13,14). The third generation inhibitors anastrozole, letrozole, and exemestane are now used clinically in breast cancer therapy.

Our research focuses on examination of the role of steroid hormones and estrogen biotransformations in breast cancer etiology. An estimated 60-70% of human breast cancers are associated with sex hormone exposure. Our hypothesis is that alterations in the breast cancer tissue microenvironment, such as exposure to exogenous agents, can influence the extent of estrogen biosynthesis and metabolism, result in altered levels of hormonally active estrogens and their metabolites, and therefore influence reproductive processes and/or breast tumor development and growth. Biochemical examination of this hypothesis in vitro has been performed in human breast cancer cell systems currently in use in our laboratories.

Flavonoid and isoflavonoid phytoestrogens exhibit numerous biochemical activities. Some natural flavones have demonstrated inhibitory activity; however, their effects on estrogen biosynthesis have not been fully examined. Investigations are underway examining the regulation of aromatase by exogenous agents such as drugs and environmental agents. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in flavonoid and isoflavonoid natural products that have weak aromatase inhibitory activity. Molecular modeling studies indicate that the low molecular weight, fairly rigid benzopyranone nucleus is easily accommodated in the binding site of aromatase. Our medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors (15). Several compounds in the initial combinatorial libraries have demonstrated good to moderate aromatase inhibitory activity in screening assays, with several compounds exhibiting approximately 30% - 70% inhibition at concentrations of 1 mM. The design, synthesis, and screening of substituted benzopyrone combinatorial libraries will allow us to harvest the biological potential of these molecules and develop more selective agents for molecular targets in breast cancer.

In addition, the effects of flavonoid and isoflavonoid phytoestrogens on estrogen metabolism have not been fully examined. MCF-7 cells (hormone-dependent) and MBA-MB-231 cells (hormone-independent) were treated with genistein (100 nM) for 5 days and then incubated with radiolabelled estradiol (100 nM, 2.5 mCi) for 0 hr., 24 hr. or 48hr. Media were extracted with ethyl acetate, and the organic residues analyzed by reverse-phase HPLC with a radioactivity flow detector. The major metabolite formed in all cases is estrone, although differences were observed between the cell lines and the various drug treatments. The formation of estrone in untreated MCF-7 cells (approximately 8.8% of radioactivity at 24 hr.) is relatively limited in contrast to untreated MDA-MB-231 cells, which about 31.5% of radioactivity at 24 hr. Treatment of MCF-7 cells with 100 nM genistein increased the conversion of estradiol to estrone up to 16.7 % in 24 hrs. The effect of genistein on estrone formation in MDA-MB-231 cells was even greater, resulting in 36.8% of the radioactivity being estrone. Thus, genistein treatment of breast cancer cells resulted in increased 17b-hydroxysteroid dehydrogenase activity and elevated formation of estrone. Increased levels of oxidative 17b-hydroxysteroid dehydrogenase activity (Type II) were confirmed by western blots. Therefore, exposure of breast cancer cells to genistein results in elevated conversion of estradiol to estrogenically weaker or inactive metabolites. This research is supported by NCI Grant R01 CA73698 and DOD Grant DAMD 17-00-1-0388.

  1. Simpson, E. R. et al., Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 15: 342-55 (1994).
  2. Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Corbin, C. J. & Mendelson, C. R., Tissue-specific promoters regulate aromatase cytochrome P450 expression. J Steroid Biochem Mol Biol 44: 321-30 (1993).
  3. James, V.H.T., McNeill, J.M., Lai, L.C., Newton, C.J., Braunsberg, H., Ghilchik, M. and Reed, M.J., Aromatase activity in normal breast and breast tumor tissues: in vivo and in vitro studies. Steroids 50: 269 (1987).
  4. Miller, W.R. and O'Neill, J., The importance of local synthesis of estrogen within the breast. Steroids 50: 537 (1987).
  5. Reed, M.J., Owen, A.M., Lai, L.C., Coldham, N.G., Ghilchik, M.W., Shaikh, N.A. and James, V.H.T., In situ oestrone synthesis in normal breast and breast tumour tissues: effect of treatment with 4-hydroxy-androstenedione. Int. J. Cancer 44: 233 (1989).
  6. Sourdaine, P., Mullen, P., White, R., Telford, J., Parker, M.G., and Miller, W.R., Aromatase activity and CYP19 gene expression in breast cancers. J Steroid Biochem Mol Biol 59: 191-198 (1996).
  7. Reed, M.J., Topping, L., Coldham, N.G., Purohit, A., Ghilchik, M.W., and James, V.H.T., Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors. J. Steroid Biochem. Mol. Biol. 44: 589 (1993).
  8. Reed, M.J., The role of aromatase in breast tumors. Breast Canc. Res. Treat. 30: 7 (1994).
  9. Brueggemeier, R.W. and Katlic, N.E. Effects of the aromatase inhibitor 7a-(4'-amino)thiophenyl-4-androstene-3,17-dione in human mammary carcinoma cell culture. Cancer Res. 47: 4548 (1987).
  10. Brueggemeier, R.W. and Katlic, N.E. Effects of an enzyme-activated irreversible aromatase inhibitor on human carcinoma cells in culture. Cancer Res. 50: 3652 (1990).
  11. Burak, Jr., W.E., A.L. Quinn, W.B. Farrar, and R.W. Brueggemeier, Androgens influence estrogen-induced responses in human breast carcinoma cells through cytochrome P450 aromatase. Breast Cancer Res. Treat. 44: 57-64 (1997).
  12. Brueggemeier, R.W., Quinn, A.L. Parrett, M.L., Joarder, F.S., Harris, R.E. and Robertson, F.M. Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens. Cancer Letters 140: 27-35 (1999).
  13. Brueggemeier, R.W. Aromatase Inhibitors -- Mechanisms of Steroidal Inhibitors, Breast Canc. Res. Treat. 30: 31-42 (1994).
  14. O'Reilly, J.M. and Brueggemeier, R.W. Development of steroidal and nonsteroidal inhibitors of aromatase for the treatment of hormone-dependent breast cancer. Current Med. Chem. 3: 11-22 (1996).
  15. Bhat, A.S. Whetstone, J.L. and Brueggemeier, R.W. Novel synthetic routes suitable for constructing benzopyrone combinatorial libraries. Tetrahedron Letters 40: 2469-2472 (1999).