Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17b-hydroxysteroid dehydrogenases (17b-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17b-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17b-HSD, 17b-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17b-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17b-HSD1, using a novel murine model. 17b-HSD1 and 17b-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17b-HSD12 was highly expressed in breast cancer cell lines, only 17b-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 lg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17b-HSD, 17b-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17b-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectinbinding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.The aggregating proteoglycans aggrecan, versican, neurocan, and brevican form the lectican (1) or hyalectan (2) family and are major components of the extracellular matrix (ECM) 1 with important functions in many tissues. The core proteins of these proteoglycans have extended central glycosaminoglycan attachment regions of varying length that are flanked by globular domains (3-6). In the cartilage proteoglycan aggrecan, the large extent of glycosaminoglycan side chain substitution and the resulting fixed charge density attracts counter-ions and water through osmotic processes. The resulting swelling pressure is crucial for the biomechanical properties of this tissue (7). The conserved N-terminal globular G1 domains anchor these proteoglycans to hyaluronan in an interaction stabilized by the link protein (8 -12). Aggrecan contains an additional globular G2 domain of unknown function between the G1 domain and the glycosaminoglycan attachment region (13). The C-terminal G3 domain is highly conserved and found in all four of these proteoglycans.We have shown previously that the G3 domain mediates binding to other ECM molecules, e.g. tenascin-R (14, 15), fibulin-1 (16), fibulin-2 (17), and fibrillin-1 (18). The G3 domain also binds sulfated glycolipids on the cell surface (19). In addition, neurocan has been reported to bind to tenascin-C (20). The ECM protein ligands for the G3 domains are all dimeric or multimeric proteins, and we have shown that they can crosslink proteoglycans from different hyaluronan/proteoglycan aggregates (17). This may well be of functional importance for the organi...

The mechanism, by which transhydrogenase couples transfer of H-equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichiu coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP' or NADPH. This indicates that the rates of release of NADP' and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP' and NADPH are accompanied by the release of H' from the enzyme and (b) increased ionic strength decreases the value of the pK, of the group responsible for H' release. Modification of the enzyme with N,Wdicyclohexylcarbodiimide led to inhibition of the rate of release of NADP' and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD', NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates.It is considered that the binding and release of H' , which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Here we report the syntheses and biological evaluation of novel inhibitors based on the estrone or estradiol template. These have been investigated by modification at the 6, 16 or 17 positions or combinations of these in order to gain information about structure-activity relationships by probing different areas in the enzyme active site. Activity data have been incorporated into a QSAR with predictive power, and the X-ray crystal structures of compounds 15 and 16c have been determined. Compound 15 has an IC50 of 320 nM for 17beta-HSD1 and is selective for 17beta-HSD1 over 17beta-HSD2. Three libraries of amides are also reported that led to the identification of inhibitors 19e and 20a, which have IC50 values of 510 and 380 nM respectively, and 20 h which, having an IC50 value of 37 nM, is the most potent inhibitor of 17beta-HSD1 reported to date. These amides are also selective for 17beta-HSD1 over 17beta-HSD2.

Abstract17b-Hydroxysteroid dehydrogenases (17b-HSDs) are enzymes that are responsible for reduction or oxidation of hormones, fatty acids and bile acids in vivo, regulating the amount of the active form that is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen 17b-HSDs have been identified to date, and with one exception, 17b-HSD type 5 (17b-HSD5), an aldo-keto reductase, they are all short-chain dehydrogenases/reductases, although overall homology between the enzymes is low. Although named as 17b-HSDs, reflecting the major redox activity at the 17b-position of the steroid, the activities of these 15 enzymes vary, with several of the 17b-HSDs able to reduce and/or oxidise multiple substrates at various positions. These activities are involved in the progression of a number of diseases, including those related to steroid metabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those of aromatase and steroid sulphatase, the development of inhibitors of 17b-HSDs is at a relatively early stage, as at present none have yet reached clinical trials. However, many groups are now working on inhibitors specific for several of these enzymes for the treatment of steroid-dependent diseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in in vivo disease models. In this review, the recent advances in the validation of these enzymes as targets for the treatment of these diseases, with emphasis on 17b-HSD1, 3 and 5, the development of specific inhibitors, the models used for their evaluation, and their progress towards the clinic will be discussed.

Type 1 17β-HSD increases 17β-estradiol exposure in grade 1 EC, thus supporting tumor growth. This enzyme represents a potential therapeutic target.

The bis -sulfamoylated derivative of 2-methoxyestradiol (2-MeOE2), 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE), has shown potent antiproliferative and antiangiogenic activity in vitro and inhibits tumor growth in vivo. 2-MeOE2bisMATE is bioavailable, in contrast to 2-MeOE2 that has poor bioavailability. In this study, we have examined the role of 17B-hydroxysteroid dehydrogenase (17B-HSD) type 2 in the metabolism of 2-MeOE2. In MDA-MB-231 cells, which express high levels of 17B-HSD type 2, and in MCF-7 cells transfected with 17B-HSD type 2, highperformance liquid chromatography analysis showed that a significant proportion of 2-MeOE2 was metabolized to inactive 2-methoxyestrone. Furthermore, MCF-7 cells transfected with 17B-HSD type 2 were protected from the cytotoxic effects of 2-MeOE2. In contrast, no significant metabolism of 2-MeOE2bisMATE was detected in transfected cells and 17B-HSD type 2 transfection did not offer protection against 2-MeOE2bisMATE cytotoxicity. This study may go some way to explaining the poor bioavailability of 2-MeOE2, as the gastrointestinal mucosa expresses high levels of 17B-HSD type 2. In addition, this study shows the value of synthesizing sulfamoylated derivatives of 2-MeOE2 with C17-position modifications as these compounds have improved bioavailability and potency both in vitro and in vivo. (Cancer Res 2006; 66(1): 324-30)

Purpose:The aim of these studies was to characterize the action of STX140 in a P-glycoproteinô verexpressing tumor cell line both in vitro and in vivo. In addition, its efficacy was determined against xenografts derived from patients who failed docetaxel therapy. Experimental Design:The effects of STX140,Taxol, and 2-methoxyestradiol (2-MeOE2) on cell proliferation, cell cycle, and apoptosis were assessed in vitro in drug-resistant cells (MCF-7 DOX ) and the parental cell line (MCF-7 WT ). Mice bearing an MCF-7 DOX tumor on one flank and an MCF-7 WT tumor on the other flank were used to assess the in vivo efficacy. Furthermore, the responses to STX140 of three xenografts, derived from drug-resistant patients, were assessed. Results: In this study, STX140 caused cell cycle arrest, cyclin B1 induction, and subsequent apoptosis of both MCF-7 DOX and MCF-7 WT cells. Taxol and 2-MeOE2 were only active in the MCF-7 WT parental cell line. Although both STX140 and Taxol inhibited the growth of xenografts derived from MCF-7 WT cells, only STX140 inhibited the growth of tumors derived from MCF-7 DOX cells. 2-MeOE2 was ineffective at the dose tested against both tumor types. Two out of the three newly derived docetaxel-resistant xenografts, including a metastatic triple-negative tumor, responded to STX140 but not to docetaxel treatment. Conclusions: STX140 shows excellent efficacy in both MCF-7 WT and MCF-7 DOX breast cancer xenograft models, in contrast toTaxol and 2-MeOE2. The clinical potential of STX140 was further highlighted by the efficacy seen in xenografts recently derived from patients who had failed on taxane therapy.