ABSTRACT
The aim of this study on testosterone (T) metabolism in benign prostatic hyperplasia (BPH) and prostatic cancer was to compare the formation of metabolites in freshly isolated epithelial cells and in cells of long-term cultures (2 passages) and to identify the 5alpha-reductase (5alpha-R) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms responsible for metabolite formation. Androst-4-enedione (A), dihydrotestosterone (DHT) and 5alpha-androstanedione (5alpha-A) formation were measured by high-performance liquid chromatography coupled to a Flo-one HP radioactivity detector. Enzyme isoforms were studied by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). T conversion into A by 17beta-HSD, rather than reduction into DHT by 5alpha-R, was by far the predominant activity in cultured epithelial cells. The metabolic profile did not differ substantially between BPH and cancer cells. Long-term cell culture led to an increase in A formation compared with the level recorded in freshly isolated cells, with no significant incidence on the relative DHT level. According to RT-PCR results, both 5alpha-R isoforms (1 and 2) and 2 17beta-HSD isoforms (2 and 3) are present in epithelial cell cultures and in tissues. According to Northern blot analyses, the mRNAs for 5alpha-R2 and 17beta-HSD4 are expressed in tissue and those for 5alpha-R1 and types 2 and 4 17beta-HSD in isolated cell cultures. Moreover, finasteride, a specific 5alpha-R2 inhibitor, inhibits DHT and 5alpha-A formation in long-term cell culture of adenocarcinoma epithelial cells plated on Matrigel, suggesting a 5alpha-R2 expression. Thus, although 5alpha-R2 is present in freshly isolated epithelial cell cultures and in long-term epithelial cells cultured on Matrigel and predominates in prostate tissue, it is the 5alpha-R1 isoform that is preferentially expressed in epithelial cell cultures.
Subject(s)
17-Hydroxysteroid Dehydrogenases/genetics , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Adenocarcinoma/enzymology , Prostate/enzymology , Prostatic Hyperplasia/enzymology , Prostatic Neoplasms/enzymology , Adenocarcinoma/genetics , Collagen/pharmacology , Culture Media , Drug Combinations , Epithelial Cells/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Laminin/pharmacology , Male , Prostatic Neoplasms/genetics , Proteoglycans/pharmacology , RNA, Neoplasm/genetics , Testosterone/metabolism , Tumor Cells, CulturedABSTRACT
Benign meningioma tumors possess significant levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity. Two different 17 beta-HSDs were discovered in human placenta: one highly estrogen specific and using NADP+/NADPH as cofactors (type-1 17 beta-HSD), and a second one that utilizes both androgens and estrogens as substrates with NAD+/NADH (type-2 17 beta-HSD). Recently, two further human 17 beta-HSDs were isolated. A testis-specific 17 beta-HSD (type-3 17 beta-HSD) favors the reduction of delta 4-androstenedione to testosterone, and a ubiquitously expressed type-4 17 beta-HSD preferentially catalyzes the oxidation of estradiol and delta 5-androstenediol. In this study we characterize the expression levels of different types of 17 beta-HSD in a wide series of tumors. Using the Northern blotting method we show that type-1, -3, and -4 17 beta-HSDs are not detectable in meningiomas. In contrast, the type-2 17 beta-HSD RNA is present in 6 of 17 meningiomas and its abundance is directly correlated with estrogenic 17 beta-HSD activity (r2 = 0.74). The presence of type-2 17 beta-HSD is also demonstrated by in situ hybridization. RT-PCR and Western blots show that type-4 17 beta-HSD is also present, though at much lower levels. The progesterone receptor level, the epidermal growth factor receptor level, and the age of the patients are not correlated with the estrogenic 17 beta-HSD activity or type-2 17 beta-HSD mRNA expression level.
Subject(s)
17-Hydroxysteroid Dehydrogenases/metabolism , Meningeal Neoplasms/metabolism , Meningioma/metabolism , Aged , Aged, 80 and over , Female , Humans , In Situ Hybridization , Male , Middle Aged , Polymerase Chain Reaction , RNA, Messenger/metabolismABSTRACT
We compare testosterone (T) metabolism in primary cultures of epithelial cells and fibroblasts separated from benign prostate hypertrophy (BPH) and prostate cancer tissues. In all cultures, androstenedione (delta 4) formed by oxidation of T by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) represented 80% of the metabolites recovered. The amounts of 5 alpha-dihydrotestosterone (DHT), formed by reduction of T by 5 alpha-reductase (5 alpha-R), were small: 5 and 2% (BPH) and 8 and 15% (adenocarcinoma) for epithelial cells and fibroblasts, respectively. Northern blot analysis of total RNA from epithelial cells (BPH or adenocarcinoma) attributed the reductive activity to the 5 alpha-reductase type 1 isozyme and oxidative activity to the 17 beta-HSD type 2. In cancer fibroblasts, only little 17 beta-HSD type 2 mRNA was detected. The 5 alpha-reductase inhibitors, 4-MA (17 beta-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) and finasteride, inhibited DHT formation with a preferential action of 4-MA on epithelial cells (BPH or adenocarcinoma) and of finasteride on fibroblasts from adenocarcinoma. Neither inhibitor acted on delta 4 formation. On the other hand, the lipido-sterol extract of Serenoa repens (LSESr, Permixon) inhibited the formation of all the T metabolites studied [IC50 S = 40 and 200 micrograms/ml (BPH) and 90 and 70 micrograms/ml (adenocarcinoma) in epithelial cells and fibroblasts, respectively]. These results have important therapeutic implications when selecting appropriate treatment options for BPH.
Subject(s)
Fibroblasts/metabolism , Prostatic Neoplasms/metabolism , Testosterone/metabolism , 17-Hydroxysteroid Dehydrogenases/genetics , 17-Hydroxysteroid Dehydrogenases/metabolism , Adenosarcoma/metabolism , Adenosarcoma/pathology , Androstenedione/metabolism , Azasteroids/pharmacology , Blotting, Northern , Cells, Cultured , Cholestenone 5 alpha-Reductase , Chromatography, High Pressure Liquid/methods , Dihydrotestosterone/analogs & derivatives , Dihydrotestosterone/metabolism , Dihydrotestosterone/pharmacology , Enzyme Inhibitors/pharmacology , Epithelium/drug effects , Epithelium/metabolism , Epithelium/pathology , Etiocholanolone/analogs & derivatives , Etiocholanolone/metabolism , Fibroblasts/drug effects , Fibroblasts/pathology , Finasteride/pharmacology , Humans , Male , Meningioma/enzymology , Meningioma/metabolism , Meningioma/pathology , Oxidoreductases/genetics , Oxidoreductases/metabolism , Placenta/cytology , Placenta/enzymology , Placenta/metabolism , Prostatic Neoplasms/pathology , RNA/analysis , RNA, Messenger , Tumor Cells, CulturedABSTRACT
Meningioma benign tumors possess significant levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity. Two different 17 beta-HSDs have been cloned and characterized. The cytosolic 17 beta-HSD I which exclusively catalyzes the interconversion of 17 beta-estradiol (E2) and estrone (E1) preferentially uses NADP+ and NADPH as cofactors. In contrast, the mitochondrial-microsomal 17 beta-HSD II catalyzes both the estrogenic as well as the androgenic substrates of the 17 beta-HSD and uses NAD+ and NADH as cofactors. We demonstrated here that the 17 beta-HSD activity in meningioma tissue homogenate is both estrogenic and androgenic with Km values of 2.4, 0.4, 14.7, and 2.0 microM for E2, E1, testosterone (T), and delta 4-androstenedione (delta 4), respectively. NAD(+)-NADH is almost exclusively used as cofactor in this tissue. Moreover, fractionation of meningioma tissue revealed that most of the 17 beta-HSD activity is present in the mitochondrial-microsomal fraction. Although Northern blot analysis on meningiomas with a specific probe for human 17 beta-HSD I showed no band, the specific cDNA probe of human 17 beta-HSD II hybridized at the expected size of 1.5 kb, which was also present in placenta. On four different meningioma tumors, we were able to correlate 17 beta-HSD II mRNA expression to high levels of 17 beta-HSD activity. Taken together, the present data suggest that the meningioma 17 beta-HSD could be the 17 beta-HSD II.
