Loss of estrogen inactivation in colonic cancer.

Age and sex differences in the incidence of colonic cancer, together with epidemiological data on patients taking hormone replacement therapy, suggest the involvement of estrogens. Analogous to the role of aromatase in breast cancer, we postulated that steroid metabolism within the colon itself may be a crucial mechanism in regulating tissue exposure to estrogens. We have characterized expression of aromatase (responsible for converting C19 androgens to C18 estrogens) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) [responsible for interconversion of active estradiol (E2) to less potent estrone (E1)] in normal and neoplastic human colon from 24 patients undergoing tumor resection. Aromatase activity was similar in homogenates from normal mucosa, tissue adjacent to tumors, and the tumors themselves. Analysis of 17beta-HSD activity indicated that the predominant activity was oxidative (E2 to E1), and this conversion was significantly lower in colonic tumors [444 (90-1735); median (95% confidence interval) pmol/mg protein x h], compared with normal mucosa [1709 (415-13828), P < 0.001]. Northern blot analyses indicated expression of messenger RNAs (mRNAs) for the type 2 and 4 isozymes of 17beta-HSD in normal colon; messenger RNA for 17beta-HSD 4 was significantly lower in tumor tissue [0.75 +/- 0.22 (mean +/- SD) arbitrary U vs. 0.43 +/- 0.17, P < 0.01]. Studies in vitro, using three colonic cancer cell lines, indicated that there was an inverse correlation between 17beta-HSD oxidative activity and the rate of cell proliferation. In addition, E1, but not E2, was shown to significantly decrease proliferation when added exogenously to the colonic epithelial cell line, SW620 cells. Colonic mucosa can regulate estrogen hormone action in an intracrine fashion. The loss of estrogen inactivation may be an important mechanism in the pathogenesis of colonic cancer.     

Relative involvement of three 17beta-hydroxysteroid dehydrogenases (types 1, 7 and 12) in the formation of estradiol in various breast cancer cell lines using selective inhibitors

We investigated the relative involvement of three reductive 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms, namely types 1, 7 and 12, in the formation of potent estrogen estradiol (E2) in 10 human breast cancer cell lines (T-47D, MCF-7, ZR-75-1, CAMA-1, BT-20, BRC-17, BRC-31, BRC-32, BRC-36 and BRN-196) and also in 1 choriocarcinoma cell line (JEG-3) using selective inhibitors. In T-47D, BT-20 and JEG-3 cells, a 17beta-HSD1 inhibitor almost completely inhibited the formation of E2 at 1microM from 60nM of estrone (E1) (98%, 91% and 90%, respectively), whereas no significant inhibition of E2 formation was obtained using inhibitors of types 7 and 12. However, we obtained lower levels of inhibition (32%, 36% and 35% respectively using inhibitors of types 1, 7 and 12 at 10microM) in MCF-7 cells and even lower and variable levels of inhibition (15%, 23% and 18% respectively using inhibitors of types 1, 7 and 12 at 10microM) in ZR-75-1 cells. No inhibition of E2 formation was observed in CAMA-1 cells with a 17beta-HSD1 inhibitor at 1microM whereas inhibitors of types 7 and 12 inhibited 40% and 30% of E2 formation, respectively. In BRC and BRN cell lines, types 1, 7 and 12 17beta-HSDs were all involved in the formation of E2, but type 12 seemed to predominate. At 10microM, each inhibitor inhibited 10-50% of the formation of E2. Using MCF-7 and BRC-32 cell lines, a combination of the three inhibitors (3x10microM) does not fully inhibit the 17beta-HSD activity (65% and 75%). In addition to identify the relative importance of types 1, 7 and 12 17beta-HSDs in the formation of E2 in human breast cancer cell lines, our results show also a great variability between each cell line. In some cases the formation of E2 was completely inhibited, but this was not the result observed in other cell lines, suggesting the presence of another enzyme involved in the biosynthesis of E2.     

Estrogen metabolism and malignancy: analysis of the expression and function of 17beta-hydroxysteroid dehydrogenases in colonic cancer

Age and sex differences in the incidence of gastrointestinal cancers suggest the involvement of sex steroids. Post-menopausal loss of estrogen in women appears to be associated with a lower risk of colonic cancer, and studies in vitro have shown that estradiol (E2) stimulates the growth of colonic cancer cell lines. Paradoxically more recent epidemiological data have shown that hormone replacement therapy (HRT) is associated with a lower risk of colonic cancer, although this may reflect differences in the composition and route of administration of HRT regimes. The precise mechanism by which estrogens influence colonic cancer in vivo remains unclear, although E2-induced growth of colonic cancer cells in vitro appears to be dependent on estrogen receptor (ER) expression. We have previously demonstrated differential responses to E2 in pre-malignant and malignant colonic cancer cell lines, without any apparent difference in ER expression. Analogous to well documented studies in breast cancer, we have postulated that local steroid metabolism in the colon may play a key role in modulating the effects of oestrogens by determining the tissue availability of active E2. Using biopsy material we have shown that the normal colonic mucosa has a high level of 17beta-hydroxysteroid dehydrogenase (17beta-HSD)-mediated E2 metabolism. Furthermore, the predominant enzyme activity, inactivation of E2 to estrone (E1), was significantly decreased in paired tumor biopsies. The presence of 17beta-HSD activity in the colon appears to be due to expression of the type 2 and 4 isozymes of 17beta-HSD (17beta-HSD2 and 4), and expression of mRNA for the latter was shown to be significantly decreased in tumours compared to normal mucosa. Further studies have characterised the expression of 17beta-HSD2 and 4 in colonic epithelial cells and in colonic cancer cell lines, and have suggested a link between estrogen metabolism and colonic cell proliferation. Data reviewed here provide evidence for the importance of 17beta-HSD isozymes as attenuators of E2 bioavailability in the colon, and emphasise a possible role for 17beta-HSD2 and 4 in the pathogenesis of colon cancer.     

Differential expression of 17beta-hydroxysteroid dehydrogenases types 2 and 4 in human endometrial epithelial cell lines

In the endometrium two enzymes are known to convert estradiol to its inactive metabolite estrone: microsomal 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) and peroxisomal 17beta-HSD4. In order to elucidate the particular function of each of these two different enzymes, the human endometrial epithelial cell lines HEC-1-A and RL95-2 were examined with respect to the expression of 17betaHSD isozymes. They were compared with human endometrium in vivo. Non-radioactive in situ hybridization revealed both enzymes in glandular epithelial cells of human endometrium. The two cell lines were screened for mRNA expression of 17beta-HSD 1-4 by RT-PCR and Northern blot. 17beta-HSD2 and 4 could be detected by either method, 17beta-HSD1 only by RT-PCR, 17beta-HSD3 not at all. Both cell lines were proven to have no receptor for progesterone which is known as a physiological inducer of several 17beta-HSD isozymes. To study the regulation of 17beta-HSD2 and 17betaHSD4, the concentration of fetal calf serum in the cell culture media was reduced stepwise to 0.3% by dilution with a defined serum replacement. This treatment led to an inhibition of 17beta-HSD2 mRNA expression and an increase in the mRNA expression of 17beta-HSD4. Concomitantly, distinct morphological changes were observed, such as a decrease in the number and length of microvilli and a decrease in the formation of domes on top of the monolayers. The endometrial epithelial cell lines HEC-1-A and RL95-2 represent a suitable in vitro model for further studies of the differential expression of the major endometrial HSD isozymes, independent of the effect of progesterone.     

Localization of type 1 17beta-hydroxysteroid dehydrogenase mRNA and protein in syncytiotrophoblasts and invasive cytotrophoblasts in the human term villi

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto- and 17beta-hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17beta-hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17beta-HSD enzyme (E17beta-HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17beta-HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17beta-HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17beta-HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17beta-HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3beta-HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17beta-HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17beta-HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17beta-HSD mRNA.     

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.     

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.     

Steroid metabolism in breast cancer

The endocrine system and its steroids have long been thought to be instrumental in the etiology of breast cancer. A large proportion of cancerous breast tissues have been shown to express estrogen (ER), androgen (AR) and progesterone (PR) receptors. It is through these receptors that steroid hormones can exert their mitogenic effects. The local biosynthesis of estrogens is believed to play an integral part in the development of hormone-dependent breast cancer and recent studies on the use of inhibitors to block this steroid production has yielded an improvement of prognosis in breast cancer patients. Consequently, the understanding of the enzymes involved in the synthesis and metabolism of estrogens in breast cancer is paramount to treating this malignancy. This review examines the biological and clinical relevance of three key endocrine enzymes: steroid sulfatase (STS), aromatase (Arom), and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type-1. The importance of the over expression and increased activity of these enzymes in breast tissue and on breast cancer is discussed. Importantly, the intratumoral biosynthesis of estrogens is examined in detail. The effects of new inhibitors of these enzymes on the growth of hormone-dependent breast cancer will also be investigated. First and second generation STS inhibitors and third generation aromatase inhibitors are showing significant promise, whereas inhibitors for 17beta-HSD type-1 are still at an early stage. However, such endocrine therapy that is currently being explored has shown promising results for patients with hormone-dependent breast cancer.     

Placental steroids in cattle: hormones, placental growth factors or by-products of trophoblast giant cell differentiation?

The bovine placenta produces large amounts of steroids, mainly estrone (E1) and progesterone (P4). Specific features of bovine placental steroidogenesis are 1) the expression of all enzymes needed for the production of estrogens from cholesterol in the trophoblast 2) an only marginal and temporal contribution to peripheral maternal P4 levels restricted to a period between approx. days 150 - 240 of gestation 3) the predominance of sulfoconjugated over free E1 and 4) a complementary setting of steroidogenic enzymes in the two morphologically discriminable trophoblast cell types, the uninucleated trophoblast cells (UTC) and the trophoblast giant cells (TGC). In cattle so far no definite information is available on the specific biological roles of placental estrogens and P4. However, the detection of estrogen receptors and progesterone receptors in the placentomes suggests a role primarily as local regulators of caruncular growth, differentiation and functions. Inconsistent with a function as a caruncular growth factor is the strong evidence that in cattle placental estrogens enter the maternal compartment almost completely as estrone sulfate (E1S), which is not active at classical nuclear receptors. On the other hand, E1S may be converted locally to free active estrogens via the action of steroid sulfatase (StS), which has been detected in specific parts of the bovine caruncular epithelium. Alternatively or in addition, StS expression in the caruncular epithelium may serve the utilization of sulfated neutral steroid precursors (e.g. pregnenolone sulfate or cholesterol sulfate) supplied with maternal blood, thus providing free substrates for further metabolization in the adjacent trophoblast. The down-regulation of P450scc and P450c17 and the up-regulation of 3beta-HSD and aromatase during the differentiation of TGC from UTC in parallel with the up-regulation of ER beta and estrogen sulfotransferase in maturing TGC suggests a function of placental estrogens primarily as autoor intracrine regulators during this process and assigns to conjugated placental estrogens a role as inactivated by-products of TGC differentiation intended for excretion. Collectively, despite some evidence from recent studies for putative roles of placental steroids in cattle their exact functions in the bovine species remain still undefined.     

The analyses of 17beta-hydroxysteroid dehydrogenase isozymes in human endometrial hyperplasia and carcinoma.

Intratumoral metabolism and synthesis of estrogens are considered to play very important roles in the pathogenesis and development of human endometrial adenocarcinoma. The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isozymes catalyze the interconversion of estradiol (E2) and estrone and thereby serve to modulate the tissue levels of bioactive E2. To elucidate the possible involvement of this enzyme in human endometrial carcinoma, we first examined the expression of 17beta-HSD type 1 and type 2 in 20 normal cycling human endometria, 36 endometrial hyperplasia, and 46 endometrial endometrioid adenocarcinoma using immunohistochemistry, and we then studied immunoreactivity of 17beta-HSD type 2 using immunoblotting analyses, the activity of 17beta-HSD type 1 and type 2 using thin-layer chromatography and their expression using RT-PCR in endometrial endometrioid adenocarcinoma. We correlated these findings with various clinicopathological parameters to examine the biological significance of 17beta-HSDs in human endometrial disorders. 17beta-HSD type 2 immunoreactivity in normal endometrium was present in all cases of secretory phase (n = 14), but not in any endometrial mucosa of proliferative phase (n = 6). In addition, 17beta-HSD type 2 immunoreactivity was detected in 27 of 36 (75%) endometrial hyperplasia and 17 of 46 (37%) carcinoma cases. 17beta-HSD type 1 immunoreactivity was not detected in all the cases examined. In both endometrial hyperplasia and carcinoma cases there were significant positive correlations between 17beta-HSD type 2 and progesterone receptor labeling index (LI). In carcinoma cases, a significant inverse correlation was detected between 17beta-HSD type 2 immunoreactivity and age. In addition, 17beta-HSD type 2 immunoreactivity was also correlated with 17beta-HSD type 2 enzymatic activity, and semiquantitative analyses of 17beta-HSD type 2 messenger RNA. No significant correlations were detected between 17beta-HSD type 2 and estrogen receptor LI, Ki67 LI, amount of aromatase messenger RNA or histological grade. These data indicated that the expression of 17beta-HSD type 2 in hyperplastic and/or neoplastic endometrium may represent altered cellular features through hyperplastic and neoplastic transformation. However, 17beta-HSD type 2 may also play some protective and/or suppressive roles toward unopposed estrogenic effects through inactivating E2 in situ, especially in premenopausal patients.     

Estrogen biosynthesis in endometriosis: molecular basis and clinical relevance

Conversion of C(19) steroids to estrogens is catalyzed by aromatase in human ovary, placenta and extraglandular tissues such as adipose tissue, skin and the brain. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by prostaglandin E(2) (PGE(2)).( )This results in local production of estrogen, which induces PGE(2) formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient hydroxysteroid dehydrogenase (17beta-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE(2 )in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.     

Aromatase as a therapeutic target in endometriosis.

In contrast to normal endometrium, the expression of aromatase is aberrant in endometriosis and is stimulated by prostaglandin E2 (PGE2). This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. Another abnormality in endometriosis--deficient 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) type 2 expression--impairs the inactivation of estradiol (E2) to estrone (E1). These molecular aberrations collectively favor accumulation of increasing quantities of E2, and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis with an aromatase inhibitor.     

Expression of the 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the human brain

An enzyme-mediated metabolism of androgens and estrogens including 17beta-HSD activity in the brain of vertebrates was discovered approximately 30 years ago. Mainly 5alpha-reductase and aromatase have been studied in detail. Recently we could demonstrate reductive and oxidative 17beta-HSD activity as well as considerable mRNA expression of the 17beta-HSD types 3 and 4 in the human brain. In the present study, we report on 17beta-HSD type 5 mRNA expression in brain tissue of women and men. Data analysis did not reveal sex specific differences, but we determined a significantly higher mRNA concentration in the subcortical white matter (SC) than in the cerebral cortex (CX). Investigation of reductive 17beta-HSD in vitro activity with 2 microM androstenedione as the substrate revealed no sex specific differences. Testosterone formation was significantly higher in SC than in CX. Moreover, enzyme activity was significantly higher in brain tissue of adults compared to that of children.     

Estrogen synthesis by osteoblast cell lines.

Estrogens play a central role in modulating bone turnover and in the postmenopausal female are formed almost exclusively by peripheral conversion of sex steroid precursors derived from the adrenals. In this study we have demonstrated that three human osteoblastic cell lines [HOS, U20S (HTB96) and MG63] possess the enzymes necessary for estrogen synthesis and metabolism. Aromatase, estradiol 17 beta-hydroxysteroid dehydrogenase (reductive and oxidative) and estrone sulfatase activities were measured in whole cell monolayers over a 20 h period by isotopic assay techniques. Significant aromatase activity was detected in all three cell lines ranging from 1.8 +/- 0.2 fmol/20 h/10(6) cells (mean +/- S.D., n = 3) for MG63 cells to 51 +/- 1.5 fmol/20 h/10(6) cells for HOS cells. The specific aromatase inhibitor, 4-hydroxyandrostenedione (1 mumol/L) completely inhibited aromatase activity in these cells. Two of the cell lines, HOS and MG63, had significant estradiol 17 beta-hydroxysteroid dehydrogenase activity with oxidative (32.7 +/- 1.9 and 1068.4 +/- 40.2 fmol/20 h/10(6) cells respectively) predominant over reductive activity (1.6 +/- 0.4 and 38.7 +/- 1.8 fmol/20 h/10(6) cells). All three cell lines were able to hydrolyse estrone sulfate to estrone with activities ranging from 13.3 +/- 1.5 fmol/20 h/10(6) cells for U20S cells to 482.2 +/- 3.7 fmol/20 h/10(6) cells for MG63 cells. Since estrogen has been implicated as a critical factor in the modulation of bone resorption and formation, the regulation of skeletal estrogen production, particularly at the time of the menopause, is likely to be an important mechanism by which bone volume is determined in physiological and pathological states.     

Structure-activity study in the class of 6-(3'-hydroxyphenyl)naphthalenes leading to an optimization of a pharmacophore model for 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) inhibitors

17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the transformation of estrone (E1) into the most potent estrogen, estradiol (E2), which stimulates cell proliferation and decreases apoptosis. 17beta-HSD1 is often strongly overexpressed in estrogen-dependent diseases (like breast cancer and endometriosis). Thus, this over expressed enzyme is a promising novel target for the development of selective inhibitors, which could be used as drugs for the treatment of these diseases. Using a structure- and ligand-based approach, a pharmacophore model was proposed and a new class of non-steroidal inhibitors of 17beta-HSD1 was designed. Enzyme inhibition was evaluated in vitro using the human enzyme. After identification of the 6-(3'-hydroxyphenyl)-2-naphthol scaffold 1, the potency of this class of inhibitors was further improved by substitution of the 1-position of the naphthalene ring by a phenyl group (compound 18, IC(50)=20nM). Compound 18 also showed a good selectivity toward 17beta-HSD2 and the estrogen receptors alpha and beta.     

Novel, potent inhibitors of 17beta-hydroxysteroid dehydrogenase type 1

Many breast tumours are hormone-responsive and rely on estrogens for their sustained growth and development. The enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is primarily responsible for the conversion of estrone (E1) into the most potent of the human estrogens 17beta-estradiol (E2). Here we report the syntheses, inhibitory activities and docking studies for a novel series of pyrazole amides which have been discovered with the aim of probing the structure activity relationships (SAR) for such a template and of using this template to mimic the potent inhibitor 1 (Fig. 1). Amides containing an aromatic pyridyl moiety have been found to give the best inhibition, indicating that the pyridyl group interacts beneficially in the active site. This work has shown that extension from this position on the pyrazole template is well tolerated and the optimization of such systems is under investigation.     

Inhibitors of type II 17beta-hydroxysteroid dehydrogenase

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are involved in the last step of the biosynthesis of sex steroids from cholesterol. This family of steroidogenic enzymes constitutes an interesting target in the control of the concentration of estrogens and androgens. Among the isoforms of 17beta-HSD, type II preferentially catalyzes the oxidation of estradiol (E(2)), testosterone (T), dihydrotestosterone (DHT), and 20alpha-dihydroprogesterone (20alpha-DHP). Based on structure-activity relationship studies, we have developed steroidal spirolactones as inhibitors of type II 17beta-HSD using different steroid nuclei: a C18-steroid (lactones 1 and 10), an antiestrogenic nucleus (lactone 2), and a C19-steroid (lactone 28). We know these inhibitors are selective for type II 17beta-HSD as no or only weak inhibition was observed for types I and III. They also have no proliferative (androgenic) activity on androgen sensitive (AR(+)) Shionogi cells whereas their proliferative (estrogenic) activity on estrogen sensitive (ER(+)) ZR-75-1 cells depends on the nature of the steroid nucleus. Lactones 1 and 10 are weak estrogens, while lactones 2 and 28 do not exert estrogenic activity, in fact lactone 2 is an antiestrogen. Lactones 1, 2, 10 and 28 were also tested in an identical assay with a series of enzyme substrates, C19-steroid diols, and known inhibitors, for the oxidation of testosterone and estradiol into androstenedione and estrone, respectively. From this comparative study, the best inhibitors of type II 17beta-HSD (oxidase activity) were identified, but none of them were clearly more potent than the hydroxylated (reduced) forms of enzyme substrates, E2, T, and DHT. Such inhibitors remain, however, useful tools to, (1) further elucidate the role of type II 17beta-HSD, and (2) regulate the level of active estrogens, androgens and progesterone.     

The effect of obesity on the ratio of type 3 17beta-hydroxysteroid dehydrogenase mRNA to cytochrome P450 aromatase mRNA in subcutaneous abdominal and intra-abdominal adipose tissue of women

OBJECTIVES: To investigate (1) whether type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), the enzyme which catalyzes the conversion of androstenedione to testosterone in the testis, is co-expressed with P450aromatase in the preadipocytes of women, and (2) whether the relative expression of type 3 17beta-HSD and aromatase varies in subcutaneous abdominal vs intra-abdominal adipose tissue of women. SUBJECTS: Subcutaneous abdominal and intra-abdominal adipose tissue was obtained from women undergoing elective abdominal surgery (age 22-78 y, body mass index (BMI) 22.4-52.9 kg/m(2)). MEASUREMENTS: Expression of type 3 17beta-HSD in adipose cell fractions was determined using RT-PCR. Preadipocyte steroidogenesis was investigated in primary cultures using androstenedione as substrate. Messenger RNA levels for type 3 17beta-HSD and aromatase were measured in adipose tissue from the subcutaneous abdominal and intra-abdominal depots using a quantitative multiplex competitive RT-PCR assay. RESULTS: Type 3 17beta-HSD is co-expressed with aromatase in the abdominal preadipocytes of women. Cultured preadipocytes from both subcutaneous abdominal (n=5) and intra-abdominal (n=5) sites converted androstenedione to testosterone, and there was minimal conversion of androstenedione to estrone. Consistent with this, the levels of type 3 17beta-HSD mRNA were significantly higher than aromatase mRNA at both sites (P<0.05; n=8 subcutaneous abdominal, n=12 intra-abdominal adipose tissue). The ratio of levels of 17beta-HSD mRNA to aromatase mRNA in intra-abdominal adipose tissue was positively correlated with BMI (n=11, r=0.61, P<0.05) and waist circumference (n=10, r=0.65, P<0.05). The converse was found in subcutaneous abdominal adipose tissue. CONCLUSION: The intra-abdominal adipose tissue of women may be substantially androgenic, increasingly so with increasing obesity, particularly central obesity. While androgen production by this adipose tissue deposit may not contribute to circulating testosterone levels due to hepatic clearance, it may have hitherto unrecognised local effects in the intra-abdominal adipose tissue and also on the liver via the hepatic portal system. These studies suggest a mechanism linking central obesity with insulin resistance and dyslipidaemia.     

17 beta-hydroxysteroid dehydrogenases--their role in pathophysiology

17 beta-Hydroxysteroid dehydrogenases (17HSDs) regulate the biological activity of sex steroid hormones in a variety of tissues by catalyzing the interconversions between highly active steroid hormones, e.g. estradiol and testosterone, and corresponding less active hormones, estrone and androstenedione. Epidemiological and endocrine evidence indicates that estrogens play a role in the etiology of breast cancer, while androgens are involved in mechanisms controlling the growth of normal and malignant prostatic cells. Using LNCaP prostate cancer cell lines, we have developed a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition into more aggressive cells. Our data suggest that substantial changes in androgen and estrogen metabolism occur in the cells, leading to increased production of active estrogens during the process. In breast cancer, the reductive 17HSD type 1 activity is predominant in malignant cells, while the oxidative 17HSD type 2 mainly seems to be present in non-malignant breast epithelial cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach in treating estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered to be estrogen target tissues, such as the gastrointestinal tract.