Human type 2 17beta-hydroxysteroid dehydrogenase in umbilical vein and artery endothelial cells: differential inactivation of sex steroids according to the vessel type.

The human placenta produces high amounts of estradiol. 17ß-hydroxysteroid dehydrogenase type 2 (17ßHSD2) is expressed by placental endothelial cells and was proposed to regulate sex hormone levels. Previous results obtained in term placenta suggested that 17ßHSD2 expression and activity differ among umbilical cord vessels. In this study, 17ßHSD2 expression level and enzymatic activity, and estrogen receptor α and ß expression levels, were measured in endothelial cell cultures from umbilical arteries (HUAEC) and vein (HUVEC) using real-time quantitative PCR, western blot, and radiolabeled steroids. 17ßHSD2-specific activities were also measured in proximal and distal segments of freshly isolated umbilical cord arteries and vein. 17ßHSD2 mRNA level and activity were higher in HUAEC than in HUVEC. Activity was higher in umbilical arteries than in the umbilical vein. In arteries, enzymatic activity was higher near the placenta, suggesting a gradient of expression. No difference was found in ERα expression, whereas ERß was expressed at a higher level in HUAEC than in HUVEC. Expression profiles of estrogen receptors and 17ßHSD2 suggest a vessel type-specific response to estrogens. Our data support a differential modulation of biologically active sex steroid levels according to the vessel type in the foeto-placental unit, with apparent higher inactivation in the arterial system.

A useful cell system for studying the regulation of 17HSD/KSR type 2 activity and expression in ovarian epithelial cancer.

17ß-Hydroxysteroid dehydrogenase/17-ketosteroid reductase (17HSD/KSR) activity and 17HSD/KSR types 1, 2, 4, and 5 mRNA levels were characterized in ovarian cancer cell lines derived from patients unexposed to radiation or chemotherapy. Activity was at the limit of detection in TOV-112D and TOV-21G cells. Activity in OV-90 was comparable to that in human placental tissue, was predominantly microsomal and was 17HSD/KSR type 2-like in substrate specificity and inhibition patterns. In monolayers, conversion of testosterone (T) to androstenedione (A) was 12-fold greater than that of A to T. Reduction of fetal bovine serum to 0.3% in the culture medium had no effect on 17ß-HSD activity. Significant levels of type 1 and type 2 mRNAs were observed in OV-90 while only trace amounts were detected in TOV-21G. In contrast, type 4 mRNA levels were comparable for OV-90 and TOV-21G. Type 5 mRNA was detected in both cell lines but its level in OV-90 was twice that of TOV-21G. In OV-90, the type 2-like activity was predominant even though the type 5 mRNA level was 2.5-fold higher than that of the type 2. OV-90 cells may be a useful system for studying the regulation of 17HSD/KSR type 2 activity and expression in ovarian epithelial cancer.

Cyclin D1 regulates hepatic estrogen and androgen metabolism.

Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.

Steroid-converting enzymes in human ovarian carcinomas.

Anti-estrogen therapies for treating ovarian carcinoma have had mixed outcomes suggesting some tumors may be estrogen-dependent. We assayed the activity levels of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD/3-KSR) and estrone sulfatase in a series of ovarian epithelial carcinomas. 17beta-HSD activity ratios with estradiol (E(2)) and testosterone (T), and inhibition by isoform-specific inhibitors were used to estimate the contributions of 17beta-HSD isoforms. Activity levels were highest for estrone sulfatase, followed, respectively by 17beta-HSD, 3alpha-HSD/3-KSR, and 3beta-HSD. E(2)/T activity ratios varied widely between samples. A 17beta-HSD type 1 inhibition pattern was observed in 23% of the samples and a type 2 pattern in 25%. E(2) formation from estrone sulfate (E(1)S) was detected in 98% (47/48) of the samples. 17beta-HSD type 1, type 2 and type 5 mRNA was detected in matched primary tumor and metastases. Evaluation of 17beta-HSD and sulfatase activity levels, activity ratios and inhibition patterns may help predict tumor response to endocrine therapy.

Estrone sulfatase activity in patients with advanced ovarian cancer.

INTRODUCTION: We sought to identify whether the sulfatase pathway was present in ovarian cancer specimens and then to determine whether a clinical correlation existed between sulfatase activity and survival. MATERIALS AND METHODS: Enzymatic activity was assessed in advanced ovarian cancer specimens via thin layer chromatography and standardized against total protein. All enzyme activities are reported in pmol/mg protein/30 min. Kaplan Meier curves of progression-free and overall survival were constructed to compare outcomes between patients with low sulfatase activity and high sulfatase activity. Median survival rates were compared using the log-rank test for survival curves. Differences in proportions between patients with low sulfatase activity versus high sulfatase activity were compared with the z-test or chi-square analysis as appropriate. RESULTS: 37 specimens from patients with advanced stage ovarian cancer were analyzed. Enzymatic activity was detected in all specimens except one. Median progression-free survival was 23.5 months for patients with low sulfatase activity compared to 6.9 months for patients with high sulfatase activity (p=0.008). Median overall survival favored the low sulfatase group (50.8 vs. 30.6 months respectively), though statistical difference was not detected (p=0.16). No other difference in clinical characteristics between patients with high or low sulfatase activity was detected. CONCLUSIONS: Sulfatase activity is widely present in ovarian cancer specimens. Increased sulfatase activity is associated with worse progression-free survival in patients with advanced stage ovarian cancer. The sulfatase pathway is a potential therapeutic target in the treatment of ovarian cancer.

Steroid metabolism in cnidarians: insights from Nematostella vectensis.

Cnidarians occupy a key evolutionary position as a sister group to bilaterian animals. While cnidarians contain a diverse complement of steroids, sterols, and other lipid metabolites, relatively little is known of the endogenous steroid metabolism or function in cnidarian tissues. Incubations of cnidarian tissues with steroid substrates have indicated the presence of steroid metabolizing enzymes, particularly enzymes with 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. Through analysis of the genome of the starlet sea anemone, Nematostella vectensis, we identified a suite of genes in the short chain dehydrogenase/reductase (SDR) superfamily including homologs of genes that metabolize steroids in other animals. A more detailed analysis of Hsd17b4 revealed complex evolutionary relationships, apparent intron loss in several taxa, and predominantly adult expression in N. vectensis. Due to its ease of culture and available molecular tools N. vectensis is an excellent model for investigation of cnidarian steroid metabolism and gene function.

Endometrial carcinoma expresses an increased cathepsin B/D ratio.

OBJECTIVES: Cathepsins B and D belong to a family of proteases involved in tumor invasion and metastasis. As such they may function as biomarkers for the aggressiveness of a given tumor. We examined the enzymatic activity of these proteins as well as the cellular and extracellular distribution of cathepsins B and D. METHODS: 39 snap frozen tissue samples were assayed for activity fluorometrically with cathepsin-specific peptide substrates in combination with specific inhibitors. 4 groups were established: benign tissue, stage I/grade 1, stage i/grade 3, and stage IIIC/any grade. IHC staining for cathepsin B with the percentage of counterstained enzyme calculated from each specimen. RESULTS: A significantly increased level of cathepsin B activity was seen in malignant tissue specimens when compared to benign tissue. The cathepsin B/D ratio confirmed and was required to detect the significance of this distinction for each malignant group when compared to benign samples. There were no differences in cathepsin B or D expression detected between the various malignant groups. IHC staining for cathepsin B was more diffuse in the malignant tissues. CONCLUSIONS: Malignant endometrium displays increased cathepsin B activity when compared benign samples. The cathepsin B/D ratio is increased for each of the malignant groups studied when compared directly to benign endometrium. The cathepsin B/D ratio cannot be utilized to distinguish the stage or grade between any of the malignant groups studied. This ratio may serve to distinguish malignant from benign tumor samples and may be a constitutive change in the malignant transformation.

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.

Cathepsins B and D activity and activity ratios in normal ovaries, benign ovarian neoplasms, and epithelial ovarian cancer.

OBJECTIVE: Cathepsins B (CB) and D (CD) belong to a family of proteases felt to be important in tumor metastasis and invasion. It has been suggested that both enzymes play a role the progression of epithelial ovarian cancer and they have been investigated as potential biomarkers for ovarian cancer. Our objective was to determine if activity ratios of these two isoforms might enhance their usefulness as biomarkers. METHODS: Ovarian cancer cell lines and snap-frozen archived tissue samples were sonicated and cathepsin activities were assayed fluorometrically with cathepsin-specific peptide substrates in combination with specific inhibitors. Tissue specimens were divided into four groups: normal ovary, benign neoplasm, early-stage (I/II) cancer, and late-stage (III/IV) cancer. Median CB and CD activity and the ratio of CB to CD (CB/CD) were compared using the Wilcoxon rank sum test. Nonparametric Spearman correlation was used to determine associations between CA-125 and cathepsin activity. Logistic regression was used to test the association between cathepsin activity and malignancy. RESULTS: In cell lines and tissue, CD activity remained relatively constant, while CB activity varied. CB activity was greatest in cancer tissue. Elevated serum CA-125 was associated with elevations in CB activity and CB/CD but not CD activity. Elevated CB activity and CB/CD as well as increasing CA-125 and age are all associated with malignancy. Multiple logistic regression shows that CB activity and age best predict malignancy status. CONCLUSIONS: CB activity is associated with invasive ovarian neoplasm. Our results do not suggest that the ratio of activity between CB and CD provides any additional information than CB activity alone. Both tissue CB activity and CB/CD activity ratios correlate with serum levels of CA-125; however there is no correlation between CD activity and CA-125.

Inhibition of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity of human lung microsomes by genistein, daidzein, coumestrol and C(18)-, C(19)- and C(21)-hydroxysteroids and ketosteroids.

Epidemiologic data suggest a relationship between dietary intake of phytochemicals and a lower incidence of some cancers. Modulation of steroid hormone metabolism has been proposed as a basis for this effect. It has been shown that aromatase, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) are inhibited by the isoflavones, genistein and daidzein, and by coumestrol. In general, the extent of inhibition has been expressed in terms of IC50-values, which do not give information as to the pattern of inhibition, i.e., competitive, non-competitive, or mixed. Less is known of the effects of these compounds on 3alpha-HSD. The human lung is known to have a high level of 17beta-HSD and 3alpha-HSD activity. During the course of studies to characterize both activities in normal and inflamed lung and lung tumors we noted that 3alpha-HSD activity with 5alpha-DHT of microsomes from normal, adult lung was particularly susceptible to inhibition by coumestrol. To clarify the pattern of inhibition, the inhibition constants Ki and K'i were evaluated from plots of 1/v versus [I] and [S]/v versus [I]. Genistein, daidzein and coumestrol gave mixed inhibition patterns versus both 5alpha-DHT and NADH. In contrast, 5alpha-androstane-3,17-dione and 5alpha-pregnane-3,20-dione were competitive with 5alpha-DHT. NAD inhibited competitively with NADH. Our findings demonstrate that phytochemicals have the potential to inhibit 5alpha-DHT metabolism and thereby affect the androgen status of the human lung. The observation of a mixed inhibition pattern suggests these compounds bind to more than one form of the enzyme within the catalytic pathway.

Androgenic and estrogenic 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms.

17beta-Hydroxysteroid dehydrogenase/17-ketosteroid reductases (17HSD/KSR) play a key role in regulating steroid receptor occupancy in normal tissues and tumors. Although 17HSD/KSR activity has been detected in ovarian epithelial tumors, our understanding of which isoforms are present and their potential for steroid metabolism is limited. In this investigation, 17HSD/KSR activity from a series of ovarian epithelial tumors was assayed in cytosol and microsomes under conditions which differentiate between isoforms. Inhibition studies were used to further characterize the steroid specificities of isoforms in the two subcellular fractions. Activity varied widely between tumors of the same histopathologic classification. The highest levels of activity were observed in mucinous tumors. Michaelis constants, maximum velocities, estradiol-17beta/testosterone (E(2)/T) activity ratios and inhibition patterns were consistent with a predominance of microsomal 17HSD/KSR2 and cytosolic 17HSD/KSR5, isoforms reactive with both E(2) and T, with evidence of estrogenic 17HSD/KSR1 in cytosol from some samples. In tumors where activity and mRNA expression were both characterized, Northern blots, PCR and sequence analysis indicated 17HSD/KSR5 was the predominant isoform. The presence of 17HSD/KSR5, which also has both 3alpha-HSD/KSR and 20alphaHSD/KSR activity, and 17HSD/KSR2 which also has 20alpha-HSD activity, could influence not only estrogen and androgen binding but progesterone receptor occupancy, as well, in receptor-containing tumors.

Altered androgen metabolism eventually leads hepatocellular carcinoma to an impaired hormone responsiveness.

Sex steroid hormones are thought, among several other risk factors, to play a role in liver malignancies. For example, from epidemiological studies in hepatocellular carcinoma (HCC), a clear disadvantage for male sex is evident. In addition, elevated levels of serum testosterone (T) and increased T to Estradiol (E(2)) ratio have been reported to predict an increased risk of HCC for male cirrhotic patients. On the other hand, palliative treatment of liver cancer patients with anti-hormones has been widely used in the past. However, the molecular mechanism(s) underlying sex steroid action on either normal or transformed liver cells, have not yet been fully clarified, nor endocrine discriminants have been satisfactorily assessed for an adequate characterization of liver cancer tissues. In this paper, we report studies on hormonal status of human liver tissues and cells, especially focusing on androgens, to better define endocrine end-points of interest for HCC. A consistent evidence from ex vivo or in vitro systems strongly suggests that high affinity binding sites of androgens are expressed at sufficient concentrations to induce a biological response in either normal or phenotipically transformed hepatocytes; in the latter, however, high heterogeneity and/or more scattering concentrations were encountered. Further, experimental data seem to suggest that lack of response to androgens may be due to a rapid metabolic conversion of steroids by neoplastic tissues and cells. Cancer hepatocytes privilege in fact 5beta more than 5alpha metabolic pathway of androgens. This may eventually lead biologically active androgens to be transformed into less active derivatives, as it occurs for T which is massively converted (>90% at 6 h) thus hindering the whole mechanism of action of androgens.

Androgen inactivation in human lung fibroblasts: variations in levels of 17 beta-hydroxysteroid dehydrogenase type 2 and 5 alpha-reductase activity compatible with androgen inactivation.

Androgens delay lung maturation through their action on lung fibroblasts. Knowing that testosterone is secreted by the lung epithelial-like cell line A-549, we have studied the metabolism of androgens by several human lung diploid fibroblasts cell lines. No 17-ketosteroid reductase activity was detected. In contrast, testosterone was transformed mainly into androstenedione and androstanedione with no 5 alpha-dihydrotestosterone formed, indicating the presence of 17 beta- hydroxysteroid dehydrogenase (HSD) type 2 and 5 alpha-reductase activities. The eight cell lines analyzed had either a low or high 17 beta-HSD type 2 activity level. No correlation between these levels and the sex or age stage of cells was established, but Northern blot analysis of human lung RNA samples of five adult subjects revealed very similar variations between subjects in the level of 17 beta-HSD type 2 mRNA. The 5 alpha-reductase activity had a marked substrate preference for androstenedione, the product of 17 beta-HSD type 2. When tritiated testosterone was used as substrate, only barely detectable levels of 5 alpha-dihydrotestosterone were observed by HPLC in the presence of the 17 beta-HSD type 2 inhibitor EM-919. The use of unlabeled testosterone or of the antiandrogen hydroxyflutamide demonstrated that the tritiated testosterone substrate itself had no effect on levels of 5 alpha-reduction. In fact, in these cells, 5 alpha-reductase has no significant activity on testosterone, but it further converts the product of 17 beta-HSD type 2, thus playing a role with 17 beta-HSD type 2 in androgen inactivation. Because androgens delay lung maturation and surfactant synthesis by their action on lung fibroblasts, it is of particular interest to find that the steroid metabolism of these lung fibroblast cells is oriented toward androgen inactivation. Because lung fibroblasts of subjects with low 17 beta-HSD type 2 expression levels are likely to be exposed to higher levels of androgens, an allelic variation of the 17 beta-HSD-2 gene is suspected, which would result in familial incidence of respiratory distress. This is in line with reported cases of familial incidence of respiratory distress.