Dehydroepiandrosterone and estrone 17-ketosteroid reductases in MCF-7 human breast cancer cells.
The identification of several steroid-transforming enzymes within human breast cancers has led to speculation that the growth of some hormone-responsive tumors might be mediated in part by intracellularly derived estrogens. Reports that MCF-7 human breast cancer cells can transform both estrone (E1)1 to estradiol (E2) and dehydroepiandrosterone (DHEA) to the estrogenic steroid 5-androstenediol (AED), have prompted us to investigate the 17-ketosteroid reductase activities (17-KSR's) which mediate these potentially important reactions. Enzyme assays were performed by quantifying the amounts of [3H]AED or [3H]E2 former from [3H]DHEA or [3H]E1, respectively, by various subcellular preparations from MCF-7 cells under a variety of experimental conditions. DHEA 17-KSR was found to be localized exclusively within cytosol, whereas the E1 17-KSR activity appeared to be nearly equally divided between the soluble and particulate cytoplasmic subfractions. The particulate E1 17-KSR appeared capable of utilizing NADH or NADPH, whereas both the cytosolic form of this enzyme and the soluble DHEA 17-KSR activity showed a strict requirement for NADPH. Although both of the soluble 17-KSR's also showed similar pH optima, several other features suggested that they are different enzymes in MCF-7. E1 did not inhibit the conversion of DHEA to AED, and DHEA did not interfere with the transformation of E1 to E2, indicating that major differences in substrate specificity exist between the two cytosolic activities. Furthermore, DHEA 17-KSR activity within cytosol stored at -20 degrees C deteriorated almost completely over twelve weeks of storage, whereas E1 17-KSR activity remained stable. Finally, although both enzymes were found to be subject to product inhibition, AED inhibited DHEA 17-KSR competitively, whereas cytosolic E1 17-KSR activity was inhibited by E2 in noncompetitive fashion. Studies of the oxidation of E2 to E1 by MCF-7 cells showed that this transformation is catalyzed by both soluble and particulate 17-hydroxysteroid oxidases which utilize either NAD or NADP as cofactor. Having previously reported the presence of a particulate NADP(H)-linked androstenedione (AE) 17-ketosteroid oxidoreductase in MCF-7, we now suggest that at least three different enzymes, one particulate and two soluble forms, participate in the conversion of 17-ketosteroids to their hormonally active 17-hydroxysteroid derivatives within this cell line. The restricted substrate requirements of each enzyme provide a rationale for developing selective enzyme inhibitors which could provide important investigational tools and potentially effective therapeutic agents.
MacIndoe
J H
JH
Department of Internal Medicine, Veterans Administration Medical Center, Iowa City, Iowa.
Hinkhouse
M
M
Woods
G
G
D015067:17-Hydroxysteroid Dehydrogenases / Q000032:analysis* / Q000037:antagonists & inhibitors; D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000037:antagonists & inhibitors; D015114:Androstenediol / Q000378:metabolism; D001943:Breast Neoplasms / Q000473:pathology*; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000473:pathology*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis*; D009376:Neoplasms, Hormone-Dependent / Q000473:pathology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D004967:Estrogens; D009363:Neoplasm Proteins; D003687:Dehydroepiandrosterone; D015114:Androstenediol; D015067:17-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; C060892:estrone reductase