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Steroid Sulfatase: Molecular Biology, Regulation, and Inhibition

Endocrinology and Metabolic Medicine and Sterix Ltd. (M.J.R., A.P., S.P.N.), Faculty of Medicine, Imperial College, St. Mary’s Hospital, London, W2 1NY, United Kingdom; and Medicinal Chemistry and Sterix Ltd. (L.W.L.W., B.V.L.P.), Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, United Kingdom

Correspondence: Address all correspondence and requests for reprints to: Professor M. J. Reed, Endocrinology and Metabolic Medicine, Imperial College, St. Mary’s Hospital, London, W2 1NY, United Kingdom. E-mail: m.reed{at}imperial.ac.uk

	Abstract

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and therefore has a pivotal role in regulating the formation of biologically active steroids. The enzyme is widely distributed throughout the body, and its action is implicated in physiological processes and pathological conditions. The crystal structure of the enzyme has been resolved, but relatively little is known about what regulates its expression or activity. Research into the control and inhibition of this enzyme has been stimulated by its important role in supporting the growth of hormone-dependent tumors of the breast and prostate. STS is responsible for the hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate to estrone and dehydroepiandrosterone, respectively, both of which can be converted to steroids with estrogenic properties (i.e., estradiol and androstenediol) that can stimulate tumor growth. STS expression is increased in breast tumors and has prognostic significance. The role of STS in supporting tumor growth prompted the development of potent STS inhibitors. Several steroidal and nonsteroidal STS inhibitors are now available, with the irreversible type of inhibitor having a phenol sulfamate ester as its active pharmacophore. One such inhibitor, 667 COUMATE, has now entered a phase I trial in postmenopausal women with breast cancer. The skin is also an important site of STS activity, and deficiency of this enzyme is associated with X-linked ichthyosis. STS may also be involved in regulating part of the immune response and some aspects of cognitive function. The development of potent STS inhibitors will allow investigation of the role of this enzyme in physiological and pathological processes.

I. Introduction

II. Molecular Biology of STS

III. Localization of STS

A. Immunocytochemical localization of STS

B. Biochemical localization of STS

IV. Regulation of STS Activity

A. Cytokines and growth factors

B. Steroids

V. Biological Roles of STS

A. In hormone-dependent breast cancer

B. STS in skin

C. STS in the immune system

D. STS, neurofunction, and memory

E. STS in reproductive tract tissues

F. STS activity in osteoblast cells

G. STS activity in leukocytes and thrombocytes

VI. Tissue Availability of Steroid Sulfates

VII. STS Inhibitors

A. Alternative substrates

B. Reversible inhibitors

C. Irreversible inhibitors

VIII. Active Pharmacophore Required for Potent Inhibition

IX. In Vivo Activity of STS Inhibitors and Efficacy in Tumor Models

X. Dual-Function Inhibitors

A. Dual aromatase-sulfatase inhibitors

B. STS and antiangiogenic microtubule disruptors

C. STS and CA

XI. Mechanism of Steroid Sulfate Hydrolysis and STS Inhibition

XII. Future Perspectives

	I. Introduction

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
AFTER MORE THAN a decade’s research to develop potent steroid sulfatase (STS) inhibitors, at least one inhibitor has now entered clinical trials to test its efficacy in postmenopausal women with breast cancer. It is therefore timely to review the role that this enzyme has in physiological and pathological conditions and to examine the rapid progress that has recently been made in developing potent STS inhibitors. STS (EC 3.1.6.2, aryl sulfatase C) is the enzyme responsible for the hydrolysis of alkyl [e.g., dehydroepiandrosterone (DHEA) sulfate (DHEAS)] and aryl steroid sulfates [e.g., estrone sulfate (E1S)] to their unconjugated forms. E1S was one of the first steroid conjugates to be isolated from the urine of pregnant mares in 1938 (1). In the past, steroid sulfates were generally considered to be end products of metabolism with their water solubility aiding excretion. However, during the last decade there has been a resurgence of interest in the roles that steroid sulfates, such as DHEAS and E1S, may have as precursors for the formation of biologically active hormones.

A major impetus to the development of STS inhibitors was to identify new drugs for use in the treatment of hormone-dependent breast cancer. These tumors in postmenopausal women are initially treated with endocrine therapy, such as antiestrogens or, more recently, aromatase inhibitors. Many breast tumors will either fail to respond to such therapies or progress after a relatively short period of time, making it necessary to continue the search for new effective therapeutic agents. While the search for STS inhibitors was in progress, it became apparent that they may also have therapeutic applications in a number of other, nononcological conditions, including regulation of part of the immune response, dermatology, and cognitive function. In this paper, we review the recent advances that have been made in understanding the molecular biology and structure of the STS enzyme. The roles that STS may have in regulating the formation of biologically active hormones are also considered. The research leading to the development of potent STS inhibitors is discussed together with the potential therapeutic importance of this new class of drug.

	II. Molecular Biology of STS

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
STS is a member of a superfamily of 12 different mammalian sulfatases (2, 3). The gene for the human STS is located on the distal short arm of the X-chromosome and maps to Xp22.3-Xpter; the gene is pseudoautosomal and escapes X-inactivation. On the Y-chromosome, there is a pseudogene for STS, which is transcriptionally inactive as the promoter, and several exons have been deleted. Sequence divergence has produced numerous stop codons in this pseudogene, and there are several large insertions. The extent of sequence similarity between the two genes suggests that they have been diverging for approximately 40 million years (4).

The locus for the human STS gene on the X-chromo-some has been cloned, characterized, and sequenced (4) (GenBank accession no. M23945; Ensembl accession no. ENSG00000101846). The structure of the gene is shown in Fig. 1. The gene consists of 10 exons and spans 146 kb, with the intron sizes ranging from 102 bp up to 35 kb. Variable mRNA transcripts, detected by Northern blotting, are due to the use of alternative polyadenylation sites within exon 10 and are not thought to be caused by splice variants (3). The cDNA for STS has been cloned and sequenced (5, 6) (GenBank accession no. M16505 and J04964). It encodes a protein of 583 amino acids, with a signal peptide of 21–23 peptides and four potential glycosylation sites of which at least two are used, at asparagine residues 47 and 259.

View larger version (11K): [in this window] [in a new window]   FIG. 1. The exon-intron structure of the human STS gene. [Derived from Ref. 4 .]

So far, there has only been a very limited study of the molecular regulation of STS. The cytokines TNF and IL-6 both up-regulate STS enzyme activity in MCF-7 breast cancer cells. However, upon further investigation, this up-regulation appeared to be posttranslationally mediated rather than occurring via any changes in gene transcription or mRNA stability (13). The promoter region of the STS gene has been characterized, and some potential tissue-specific regulatory elements have been identified (14). The promoter is unusual, because it resembles neither a housekeeping gene nor a tightly regulated gene. It lacks a TATA box, is not GC rich, and lacks binding sites for Sp1 and other known transcription factors. The transcription start sites were mapped by primer extension and S1 nuclease protection assays. The major start site is at –221 with respect to the A nucleotide of the initiating methionine, with other minor transcription start sites mapped to –197, –206, and –241. The basal promoter region was identified as a 110-bp region from –192 to –302 using transient transfection reporter gene assays. Four other upstream regulatory elements (UREs) were identified: URE1 –305 to –572, URE2 –870 to –1086, and URE3 –1087 to –1253, which all act as enhancers. The three enhancer regions are counterbalanced by the presence of a negative regulatory region at –1253 to –1458. The basic promoter and URE activities could be detected only in the human choriocarcinoma JEG-3 cells, which are of placental origin and have high STS activity. Transfections of the basic promoter and UREs into COS-1, HeLa, and B82 cells gave no activity, suggesting that tissue-specific factors are required for activity of the STS promoter.

	III. Localization of STS

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
STS activity was first demonstrated in rat liver microsomes by Dodgson et al. (15). Since then, it has been found in testis, ovary, adrenal glands, placenta, prostate, skin, brain, fetal lung, viscera, endometrium, peripheral blood lymphocytes, aorta, kidney, and bone. It is believed to be virtually ubiquitous in small quantities. The organ and tissue distribution varies considerably between different mammals. It is reported to be absent in the guinea pig and some marsupial livers and is undetectable in erythrocytes. The richest source of STS is the placenta. STS has been detected in various tissues by 1) immunohistochemistry, 2) biochemical analysis of hydrolytic products of various sulfated substrates (by colorimetric, fluorimetric, or radiometric methods), and 3) more recently in combination with mRNA expression levels using RT-PCR.

A. Immunocytochemical localization of STS
Using an azo-coupling histochemical method, Partanen (16) was unable to demonstrate the presence of STS in the epithelium of ducts and lobules of the normal breast, although activity was detectable in some samples of benign and malignant breast tissues. With the availability of purified preparations of STS (particularly human placental STS), anti-STS (polyclonal and monoclonal) antibodies were obtained, and specific immunohistochemical methods were developed to examine the subcellular localization of STS. In cultured human skin fibroblasts, STS was localized on the rough endoplasmic reticulum, Golgi cisternal, trans-Golgi reticulum, and, to a lesser extent, in plasma membranes and components of the endocytic pathway (i.e., coated pits, endosomes, and multivesticular endosomes). No STS immunostaining was detected in lysosomes (17). Immunohistochemistry with a monoclonal antibody to placental STS, combined with electron microscopy, also localized STS to the membranes of the endoplasmic reticulum, the nuclear envelope in rat hepatocytes, the proximal tubules in the kidney, and in the pineal gland, choroid plexus, and adenohypophysis of the rat brain (18). More recently, immunohistochemical evidence for the presence of STS has been obtained in the cytoplasm of ovarian clear cell adenocarcinomas (19), in glandular epithelial cells of the basilar layer of the endometrium but not the myometrium (20), and in vascular smooth muscle cells from the aorta (21). Immunohistochemistry has also been combined with RT-PCR to examine the localization and expression of STS in human fallopian tubes (22). STS was found to be localized in the secretory cells of fallopian tubes, and a higher number of positive cells were found in tissues obtained during the early luteal phase than in tissues collected during the follicular phase of the menstrual cycle. In agreement with these findings, abundant expression of STS mRNA was found in tissues from the early luteal phase (22). In another study, STS mRNA, enzyme activity, and immunoreactivity were assessed in normal human adult and fetal tissues (23). Amplified STS mRNA transcripts were weakly expressed in adult lung, aorta, liver, thyroid, testis, uterus, and all fetal tissues examined. Relatively high levels of STS activity were found in adult liver and the adrenal gland. The highest activity was detected in the placenta but, in keeping with the lower sensitivity of this technique, STS immunoreactivity was detected only in placental syncytiotrophoblasts. The same researchers detected STS immunoreactivity in breast carcinoma cells in 74% of cases, and this was significantly associated with its mRNA level and enzyme activity (24). An affinity-purified monoclonal antibody (KW 1049), raised against STS purified from human placenta that did not cross-react with arylsulfatases A or B, was used for the investigations in normal and malignant human tissues (23, 24).

B. Biochemical localization of STS
Historically, STS has been detected in microsomes or whole-tissue homogenates using biochemical or radiometric assays of substrate hydrolysis. For specific measurements of STS activity, [6,7-³H]E1S or [7-³H]DHEAS are used in buffer at pH 7.4 [based on Burstein and Dorfman (25)]. Phosphate buffer is preferred because it completely inhibits arylsulfatases A and B. The activities of both of these enzymes are relatively low at pH 7.4, which further improves the specificity of the assay. These assays have been used to identify and characterize STS activities in human leukocytes (26), brain (27, 28), osteoblast cell lines (29, 30), ovarian granulosa cells (31, 32), and rat testis (33).

The central role of placental STS for the formation of estriol in the fetoplacental unit, its abundance in the placenta, and the virtual absence of detectable activity in cases of the inherited disorder of placental STS deficiency and recessive X-LI have led to the enzyme from human placenta being extensively investigated. Human placental STS has been purified to homogeneity and has been well characterized. Depending on the extent of glycosylation, the purified STS has a molecular mass of approximately 65 kDa (6). Whereas evidence from early investigations suggested that aryl sulfatase C and STS may have been different enzymes, biochemical and genetic analyses have confirmed that there is only one enzyme. Chromatography of placental microsomal extracts has revealed that both activities colocalize in the same fractions (34, 35, 36). Purified STS hydrolyzes aryl sulfates (e.g., p-nitrophenyl-sulfate, E1S) as well as alkyl sulfates (DHEAS, pregnenolone sulfate, deoxycorticosterone sulfate, cholesterol sulfate), and, to a lesser extent, iodothyronine sulfates (37, 38, 39). In addition, since the first observation by Jobsis et al. (40), that sons of women with sulfatase-deficient placentas develop X-LI, the link between the deficiency of microsomal STS and X-LI has been confirmed by several groups. In these subjects, enzyme activity toward both aryl- and alkyl-steroid sulfates was lacking in all tissues examined. In keeping with the lack of STS activity, plasma concentrations of all steroid sulfates are elevated. In subjects with STS deficiency, the activities of aryl sulfatases A and B are normal (41, 42, 43). Furthermore, when the cDNA for human placental STS was transfected into COS-1 cells, the expressed protein hydrolyzed aryl (E1S) and alkyl (DHEAS) steroid sulfates, with the hydrolysis of both substrates being blocked by a single inhibitor (44). Although there is only one gene for STS, some evidence has emerged that different isoforms of the enzyme may exist in rodents and humans. After the observation of Nelson et al. (45) that two isoforms may exist in mice, two isoforms (microsomal and nuclear) were shown to exist in rat liver and human placenta (46, 47). In humans, two isoforms (slow and fast) were identified in fibroblasts (48, 49, 50). It is possible that these isoforms are the result of posttranslational modifications. Hence, it is apparent from biochemical and immunohistochemical localization studies that STS is found mainly in target tissues of the reproductive tract (i.e., endometrium, ovarian, prostate, testis, placenta), the breast, skin, brain, bone, and blood. The biological role of STS in these tissues/organs is discussed in Section V.

	IV. Regulation of STS Activity

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
The action of STS makes a major contribution to in situ estrogen production in hormone-dependent malignant tissues. Although expression of STS mRNA and STS activity is increased in malignant breast (and endometrial) tissues compared with nonmalignant tissues, little is known about the regulation of its expression or activity. Because the expression of other enzymes of steroidogenesis (such as aromatase) is known to be regulated by cytokines, growth factors, steroids, and prostaglandin E2, some of these factors have also been tested to assess whether they would induce STS.

A. Cytokines and growth factors
The cytokines IL-6 and TNF act synergistically to increase STS activity in breast cancer cells (51, 52). Furthermore, these cytokines increase STS activity without the use of promoter/enhancer elements, suggesting that the control of STS activity is via posttranslational modification of cysteine to formyl glycine in the active site or indirectly via changes in membrane fluidity or organic anion transporters, allowing increased uptake of the hydrophilic substrate (13). In contrast, the inflammatory cytokine IL-1ß decreases the activity and expression of STS mRNA in human endometrial stromal cells, in a dose-dependent manner, and this effect is antagonized by the IL-1 receptor antagonist (53). IL-1ß also suppressed STS activity and mRNA expression in vascular smooth muscle cells derived from human aortas (21). The presence of these cytokines in breast cyst fluid may explain the differential regulation of STS in breast cancer cell lines by breast cyst fluid (54). In a separate study, both basic fibroblast growth factor and IGF-I were found to increase STS activity in a dose- and time-dependent manner in MCF-7 and MDA-MB-231 breast cancer cells. This induction was inhibited by cycloheximide, indicating the requirement for new protein synthesis (55). These growth factors, which are thought to be secreted by breast tumors, may therefore increase local production of estrogens.

B. Steroids
Schneider et al. (56) first reported that in utero androgen exposure is required for induction of androgen-responsive hepatic STS in male rats. Lam and Polani (57) used exogenous testosterone treatment and concluded that STS induction is, in part, controlled by the male hormones in the mouse. Moutaouakkil et al. (58) observed that STS was highest in the uteri of pregnant guinea pigs compared with that in the uteri of fetal, castrated, or mature females, suggesting estrogenic regulation. The possibility of substrate induction of in vivo STS activity in liver and white blood cells in ovariectomized rats was confirmed by administration of exogenous E1S to ovariectomized rats (59). In contrast, a decrease in STS mRNA levels was found when MCF-7 breast cancer cells were treated with the progestagen Promegestone (R-5020) (60). However, exposure of MCF-7 and MDA-MB-231 breast cancer cells to the progestagen, medroxyprogesterone acetate, stimulated STS activity in these cells (61). Because medroxyprogesterone acetate is known to affect membrane fluidity, the enhanced STS activity might be explained by increased substrate availability from the medium. In addition, the availability of sulfated substrates may be increased by the induction of specific high-affinity transporters. It has also been reported that progesterone increased the uptake of inorganic sulfate in endometrial epithelial cells through induction of a high-affinity transport system (62). Whether progesterone or other steroids induce specific transporters for sulfated steroids in endometrial and/or other tissues remains to be explored. Recently, retinoids and 1,25-dihydroxy vitamin D₃ have been reported to induce STS activity and expression in HL-60 promyelocytic cells (63). However, the molecular mechanisms underlying cytokine or steroid induction of STS activity and/or expression remains to be explored. Furthermore, factors governing the extent of posttranslational modification of cysteine-formyl glycine, glycosylation, and translocation to the endoplasmic reticulum are all likely to influence the activity of STS.

	V. Biological Roles of STS

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
A. In hormone-dependent breast cancer
1. Hydrolysis of E1S.
Estrogens have a major role in supporting the development and growth of tumors in hormone-dependent tissues such as the breast and endometrium (64, 65). The highest incidence of breast cancer occurs in postmenopausal women after cessation of ovarian production of estrogens. However, estrogens continue to be produced in postmenopausal women by the peripheral conversion of androstenedione (Adione) to estrone (E1), a reaction mediated by the aromatase enzyme complex (66, 67). In postmenopausal women, the production rates for E1 and estradiol (E2) are approximately 40 µg/24 h and 6 µg/24 h, respectively (68). Much of the estrogens that are formed can be converted to estrogen sulfates by the actions of estrone sulfotransferase and phenol sulfotransferase (69, 70, 71, 72). Sulfation of estrogens changes them from being hydrophobic to hydrophilic molecules. In addition, because estrogen sulfates are unable to bind to the estrogen receptor (ER), they are biologically inactive. Circulating concentrations of E1S are much higher than that of the unconjugated estrogens (73, 74). Estrogen sulfates bind to albumin and have a prolonged half-life in blood (up to 9 h) compared with the much shorter half-lives of E1 and E2 (75). The high circulating concentrations of E1S together with its prolonged half-life have given rise to the view that E1S may act as a reservoir for the formation of biologically active estrogens via the action of STS (76, 77, 78, 79).

In contrast to the low circulating levels of E1 and E2 in postmenopausal women, there is now general agreement that their concentrations are much higher in normal and malignant breast tissues (80, 81). Concentrations of E1 and E2 in malignant breast tissues can be up to 10-fold higher than those found in plasma. There is also evidence for high levels of E1S and estradiol sulfate (E2S) in breast tumors (74). Surprisingly, although plasma estrogen concentrations in postmenopausal women are much lower than in premenopausal women, breast tumor estrogen levels are similar in both groups of women (82, 83). The origin of estrogens in breast tumors has been the subject of intensive research during the last decade. There are two possible mechanisms that could account for this: 1) uptake from the circulation and binding with high affinity to ERs, or 2) in situ synthesis from estrogen precursors. Although uptake and binding to ERs may make an important contribution to tissue estrogen concentrations, the finding that levels are similar in ER-positive (ER+) and ER-negative (ER–) tumors suggests that local synthesis makes a major contribution to breast tumor estrogen concentrations (84, 85).

Three enzyme systems are required for the formation of E2 from androgen precursors in breast tissues and include the aromatase, which converts Adione to E1 and 17ß-hydroxysteroid dehydrogenase (17ßHSD) type 1, which reduces E1 to E2, the biologically active estrogen that interacts with the ER. In addition, STS can act on E1S, formed as a result of sulfotransferase activity, to form E1, which can subsequently be converted to E2 (Fig. 2). All of these enzymes have been identified in malignant breast and endometrial tissues (86, 87). However, whereas aromatase activity is detected in only 40–60% of breast tumors, STS activity is present in most breast tumors (86, 88). Furthermore, the activity of STS is considerably higher than that of the aromatase enzyme in breast tumors (86). Using the appropriate substrate concentrations, it was found that as much as 10-fold more E1 could originate from E1S, via the sulfatase pathway, than from Adione by the aromatase route (89).

View larger version (22K): [in this window] [in a new window]   FIG. 2. The origin of estrogenic steroids in postmenopausal women with hormone-dependent breast cancer. SULT, sulfotransferase; 3ß-HSD-isomerase, 3ß-hydroxysteroid dehydrogenase-C5,C4-isomerase.

STS mRNA expression was found to be an independent prognostic indicator in predicting relapse-free survival, with high levels of expression being associated with a poor prognosis (92). One possible explanation for this finding was suggested, i.e., in breast tissues expressing high levels of STS mRNA, tumor cells that escape surgical removal may grow very fast, and therefore patients may relapse earlier. Whereas previous investigations found no link between time to relapse and STS activity in breast tumors (93, 94), the original findings of Utsumi et al. (92) have now been confirmed in two further investigations (24, 95). In one study, it was found that the association between STS mRNA expression and prognosis applied only to ER+ tumors. Interestingly, high STS mRNA expression was associated with a poor prognosis in both pre- and postmenopausal women. This finding led to the suggestion that even in premenopausal women, intratumoral estrogen synthesis may play an important role in the growth of breast tumors. The role of aromatase mRNA expression analysis as a prognostic marker was also examined in view of the pivotal role that the enzyme is considered to have in regulating tumor estrogen synthesis. Aromatase mRNA expression was found to have no prognostic value, a finding consistent with previous studies that examined aromatase activity as a prognostic indicator (96, 97). The lack of prognostic value of aromatase mRNA determination led the authors to speculate that the sulfatase pathway may be more important than the aromatase route for intratumoral estrogen synthesis. STS mRNA expression was also found to correlate with tumor size and to be significantly higher in tumors with lymph node metastasis than in those without lymph node metastasis (24, 95). An examination of the intratumoral expression of genes from the estradiol metabolic pathway has provided further confirmation of the high expression and prognostic significance of STS mRNA expression (98).

Immunohistochemistry and STS mRNA expression of laser-captured microdissected samples were also used to examine the location of STS within breast tumors (24). STS immunoreactivity was detected in the cytoplasm of cancer cells (Fig. 3) with STS mRNA expression being detected in microdissected carcinoma cells but not in stromal cells. This contrasts with reports as to the localization of the aromatase enzyme. Biochemical studies have consistently revealed higher aromatase activity in the stromal rather than the epithelial component of breast tumors (99). Immunohistochemical studies, however, have provided evidence for both an epithelial and stromal location for the aromatase enzyme complex (100, 101, 102).

View larger version (118K): [in this window] [in a new window]   FIG. 3. Breast tumor showing immunohistological localization of STS in malignant epithelial cells [Reproduced with permission from: T. Suzuki et al.: Cancer Res 63:2762–2770, 2003 (24 )].

Although this infusion study clearly demonstrated that NMU-induced mammary tumors in rats can be stimulated to grow by E1S, it did not differentiate between hydrolysis of E1S occurring in peripheral tissues, such as liver, and that occurring within the tumor. Two elegant studies have addressed this question, using the NMU-induced mammary tumor model or inoculation of MCF-7 breast cancer cells transfected with the STS cDNA. In the NMU model, a double-isotope infusion technique was used to determine the extent of in situ E1 formation from E1S in the tumor (106), based on a method that had previously been employed to measure the extent of formation of E1 from Adione in human breast tumors (107). For this, ¹⁴C-labeled E1 was infused into animals over a 3-d period to ensure that an isotopic steady state had been achieved. By measuring [¹⁴C]E1 levels in tumor tissue and blood, an index of the uptake of unconjugated E1 into the tumor can be calculated. By simultaneously infusing [³H]E1S, it is possible to calculate how much E1 is being formed within the tumor. Some of the infused [³H]E1S will be hydrolyzed in peripheral tissues, with some of the released [³H]E1 being taken up by the tumor. As uptake from the circulation can be calculated from the infusion of [¹⁴C]E1, any [³H]E1 in the tumor above that expected to be present due to uptake is considered to be formed by in situ synthesis. Using this technique, it was found that as much as 50% of the E1 formed within the tumor could originate from E1S.

As an alternative approach to investigate the importance of in situ formation of unconjugated estrogen from estrogen sulfates, MCF-7 cells transfected with either a vector (MCF-7v) or vector containing the STS cDNA (MCF-7_STS) were inoculated into the flanks of ovariectomized nude mice (108). The incidence of proliferating tumors in mice bearing MCF-7_STS cells, supplemented with E2S (71%), was significantly higher than in animals bearing this cell line but not supplemented with E2S (22%). Supplementation with E2S and subsequent hepatic hydrolysis were not sufficient to stimulate the growth of MCF-7v cells. This finding demonstrates the importance of in situ estrogen synthesis, compared with that occurring in peripheral tissues, in supporting tumor growth. E2S was used for these studies because, unlike E1S, it does not require the liberated steroid to be reduced by estradiol dehydrogenase (type 1) before being biologically active.

Interestingly, results from both in vitro and in vivo experiments have suggested the possibility that estrogen sulfates may have different biological activity than their unconjugated counterparts in cells expressing high STS activity. E2S was found to be more mitogenic than E2 in vitro producing a greater increase in anchorage-independent colony formation in the MCF-7_STS clones (108). In vivo the volumes of tumors of animals supplemented with E2S (138 mm³) were greater than those in animals supplemented with E2 (51 mm³). One possible explanation for this observation is that some STS activity may reside in the nucleus (46). Evidence for a nuclear STS isozyme has been obtained, and it is possible that the formation of active estrogen by STS within the nucleus may not be subjected to the same degree of inactivation by 17ßHSD type II or sulfotransferase before exerting their action.

2. Hydrolysis of DHEAS.
Evidence for the role that DHEAS, and its unconjugated metabolite DHEA, may have in breast cancer stems from two sources. First, steroid dynamic studies have revealed that these steroids can act as precursors for the formation of steroids with estrogenic properties, such as 5-androstenediol (Adiol). Second, studies in cells and animals have revealed that DHEAS, DHEA, and Adiol can stimulate the proliferation of breast cancer cells in vitro and induced mammary tumors in vivo. DHEAS is the most abundant steroid secreted by the adrenal cortex and, like estrogen sulfates, its half-life in plasma (10–20 h) is considerably longer than that of unconjugated DHEA (1–3 h) (109, 110). Isotopic infusion studies have revealed that, in women, as much as 75% of the daily production rate of DHEAS is converted to DHEA in peripheral tissues (111). After removal of the sulfate group by STS, the resulting DHEA can undergo reduction to Adiol, a steroid of particular importance with regard to breast cancer development. In postmenopausal women, the major proportion of Adiol formed is derived in peripheral tissues from DHEAS and DHEA (112). DHEAS can also be converted to Adiol-sulfate, but the contribution that this pathway makes to Adiol production remains to be resolved. Adiol, although an androgen, can bind to the ER with a somewhat lower affinity than that of E2. However, as the plasma concentrations of Adiol are at least 100-fold higher than those of E2 in postmenopausal women, it is considered to be equipotent with E2 as an estrogen in this group of women (113).

It has been known for many years that Adiol can stimulate the growth of ER+ breast cancer cells in vitro (114, 115). In addition, in vivo studies employing 7,12-dimethylbenz[a]-anthracene-induced mammary tumors in rats revealed that Adiol could stimulate tumor growth (116). Importantly, the aromatase inhibitor 4-hydroxyandrostenedione did not block the ability of Adiol to stimulate tumor growth. This finding showed that Adiol did not need to be converted to an estrogen in order to stimulate tumor growth. More recent studies have revealed that DHEA and Adiol can directly activate the ER and stimulate the proliferation of breast cancer cells (117). Coincubation of these steroids with an aromatase inhibitor did not block their ability to activate the ER. Using a physiological concentration of DHEAS, mass spectrometry analysis has revealed that it can be converted to estrogens and Adiol in MCF-7 breast cancer cells (118).

Further evidence for an important role of adrenal androgens and the sulfatase pathway in breast cancer was obtained from a study in which their effects on MCF-7 breast cancer cell proliferation were examined (119). DHEAS, DHEA, and Adiol were all found to stimulate cell proliferation, but their ability to do so was blocked by the ER antagonist nafoxidene, but not by aromatase inhibitors. In contrast, a potent STS inhibitor completely blocked that ability of DHEAS to stimulate cell growth. These results provide strong evidence that the stimulation of cell growth by DHEAS occurs via an aromatase-independent pathway that can be blocked by a STS inhibitor.

There is, therefore, convincing evidence that adrenal androgens and their metabolites can stimulate breast cancer cell growth in vitro and induced mammary tumors in rodents. Convincing clinical evidence was obtained recently in support of a role for DHEAS in stimulating breast tumor growth in humans (120). In a study carried out to monitor serum DHEAS concentrations in women being treated with third-generation aromatase inhibitors, the important observation was made that, whereas those with stable disease had low (0.6 µM) levels of DHEAS, levels were elevated (3.8 µM) in women in whom tumor progression occurred. Serum levels of E1 and E2 in all subjects remained suppressed to minimal detectable levels. It was concluded from this study that, in patients with progressive disease, DHEAS appeared to stimulate tumor progression and led to the suggestion that this finding had serious implications for the use of aromatase inhibitors on their own. A likely explanation for this observation is that DHEAS is converted to DHEA by STS. The subsequent reduction of DHEA will yield a steroid, Adiol, for which there is now convincing evidence that it can stimulate breast cancer cell growth. Inhibition of STS, in addition to blocking the formation of E1 from E1S, should also reduce the production of Adiol, by blocking the conversion of DHEAS to DHEA (Fig. 2).

B. STS in skin
STS is also found in the epidermis, and there is increasing evidence that its action within skin may make an important contribution to androgen production in this tissue. It has been known for some time, since the description of a deficiency of STS in X-LI (121, 122), that STS has an important role in skin function. Clinically, X-LI is characterized by scaling of the skin with large, dark-brown scales and an increase in stratum corneum thickness (123). Lipids are important for normal stratum corneum structure and function and may be important for the process of normal desquamation. Concentration of cholesterol sulfate in stratum corneum, and the scales associated with X-LI, are increased (5-fold) compared with levels in stratum corneum from normal subjects (124). Because STS inhibitors currently in development could severely reduce STS activity in skin, it is reassuring to note that ichthyosis can be readily treated by the topical applications of keratolytic agents, such as ammonium lactate or cholesterol cream (125).

Plasma concentrations of DHEAS can be increased in subjects with androgenic alopecia or hirsutism (126, 127). It is therefore possible that this steroid sulfate may be an important precursor for the formation of more active steroids within the skin. DHEAS can be converted to 5-dihydrotestosterone, the androgen that activates the androgen receptor, in axillary hair follicles (128). Using an immunohistochemical technique, STS was found to be predominantly expressed in the dermal papilla of hair follicles (129, 130). STS activity was also highest in the dermal papilla fraction of hair follicles. Its activity could be effectively inhibited with 1 nM of the potent STS inhibitor estrone-3-O-sulfamate (EMATE) (130). In patients with acne vulgaris, there is some evidence of increased STS immunoreactivity in affected skin areas (131). Thus, STS inhibitors may be of value in treating skin and/or hair conditions in which the action of the enzyme may be increasing local production of biologically active androgens.

C. STS in the immune system
Although DHEAS is secreted in large amounts by the adrenal cortex, it has remained controversial as to whether it has a specific biological role apart from serving as a precursor for the formation of active androgens and estrogens. Studies by Daynes et al. (132, 133) and Rook et al. (134) have suggested that DHEAS/DHEA may have an important role in regulating T-helper (Th) cell maturation. Th cells can progress to either a Th1 or Th2 phenotype, each of which secretes a characteristic profile of cytokines (e.g., Th1 cells secrete IL-2 and interferon-; Th2 cells secrete IL-6 and IL-10). The response of Th cells is mutually exclusive, with interferon- inhibiting the formation of Th2 cells and IL-10 inhibiting the formation of Th1 cells (135, 136).

Plasma IL-6 concentrations were found to be elevated in elderly human subjects, reflecting the increased production of this cytokine by Th2 cells that occurs with aging. In aged mice, in which IL-6 plasma concentrations were also increased, it was possible to correct the elevated levels by the acute or chronic administration of DHEA or DHEAS (137). These studies also revealed that in vitro DHEA, but not DHEAS, was able to suppress the release of Th2 cytokines. Thus, STS, which is present in macrophages within the lymphoid tissues where Th cell maturation occurs and which converts DHEAS to DHEA, has a crucial role in regulating part of the immune response. From such investigations it has emerged that the balance of DHEA to glucocorticoid determines whether Th cells progress to either a Th1 or Th2 phenotype, i.e., DHEA favors development to Th1 cells whereas cortisol promotes a Th2 response.

Using a contact sensitization model, convincing evidence has been obtained that in vivo DHEA and DHEAS have an immunostimulatory role (138). However, the ability of DHEAS, but not DHEA, to act as an immunostimulant was completely blocked by the coadministration of the potent STS inhibitor EMATE. Because a number of pathological conditions, such as rheumatoid arthritis, may result from an inappropriate immune response and increased production of Th1 cytokines, inhibition of STS could be of therapeutic benefit in such conditions. Using a collagen-induced model of arthritis, evidence has been obtained showing that the progression of arthritis was markedly altered by the STS inhibitor EMATE (139).

The finding that DHEA has a role in regulating the Th1/Th2 immune response has provided an important insight into the regulation of estrogen synthesis in women (140, 141). IL-6 has a major role in regulating peripheral aromatase activity (142). It has been known for many years that the peripheral aromatase activity increases upon aging and is also higher in obese subjects (143). It is also known that levels of plasma IL-6 increase with aging and its production is increased in obese subjects (137, 144). It is well established that the production of DHEAS starts to decrease from the mid-20s (145). This reduction in the production of DHEAS will favor a Th2-type cytokine response with increased production of IL-6. Thus, increased production of IL-6 is the most likely explanation to account for the increase in aromatase activity detected in aging and obese subjects.

D. STS, neurofunction, and memory
In addition to being synthesized in the adrenal cortex, steroids such as DHEAS and DHEA are also formed in parts of the central nervous system and are therefore classified as neurosteroids (146, 147). These neurosteroids have important roles in regulating brain function. Sulfated steroids, e.g., DHEAS and pregnenolone sulfate, are considered to act as -aminobutyric acid_A receptor antagonists, whereas their unconjugated analogs act as -aminobutyric acid_A receptor agonists (148). In addition, both the sulfated and unsulfated forms of these steroids act positively to modulate N-methyl-D-aspartate receptor function (149).

Because blood levels of DHEAS and DHEA decrease with aging, experiments were performed to examine the possibility that administration of these neurosteroids to rodents could improve memory. Intracerebroventricular or sc administration of DHEAS produced significant memory-enhancing effects in mice when tested using a foot-shock active avoidance training method (150, 151). Although there is convincing evidence that increasing blood levels of DHEAS and DHEA in rodents can result in memory-enhancing effects, it is not known whether such effects result from the sulfated or nonsulfated form of the neurosteroid. Because STS activity is present in brain tissues, it is possible that DHEAS could be hydrolyzed to DHEA by the action of this enzyme (152). With the advent of potent STS inhibitors, such as EMATE, it became possible to test whether the sulfated or unsulfated form of DHEA was responsible for the memory-enhancing effects of these neurosteroids (153). DHEAS is known to reverse scopolamine-induced amnesia in rodents. Blocking the hydrolysis of DHEAS with EMATE potentiated the ability of this sulfated neurosteroid to reverse scopolamine-induced amnesia. Similar results were obtained in this model using the nonsteroidal STS inhibitor (p-O-sulfamoyl)-N-tetradecanoyl tyramine (154). These findings strongly suggest that it is the sulfated form of DHEA that is responsible for the memory-enhancing effects of this steroid in rodents. Although results from these studies suggest that STS inhibitors may have a role in modulating the neuroexcitatory effects of steroid sulfates in rodents, there is, as yet, no information as to their possible affects in humans. In subjects with sulfatase deficiency, there is no evidence to suggest any abnormality in cognitive function. This suggests that the long-term therapeutic use of STS inhibitors should not have any adverse neurological effects. Whereas decreases in blood levels of DHEA and DHEAS occur in humans with aging, there is no evidence for a decrease in rodents. Therefore, experiments in rodents employing DHEA or DHEAS must be interpreted with caution.

Inhibition of STS has recently been shown to increase aggressive behavior in CBA/H mice (155). Experimental evidence had previously indicated a possible link between attack behavior and the pseudoautosomal region of the Y-chromosome, which contains the sulfatase gene (156). The finding of a correlation between the initiation of aggressive behavior and liver STS activity in mice also suggests that the sulfatase gene could be a candidate for attack behavior in mice (157). Using a nonsteroidal inhibitor, a single oral dose was found to significantly inhibit brain STS activity and increase the effect of DHEAS on aggressive behavior in CBA/H mice (155).

E. STS in reproductive tract tissues
1. Female.
STS activity has been detected in most tissues of the female reproductive tract. It is present in ovarian tissues from pre- and postmenopausal women, suggesting that in the ovary sulfated precursors, such as DHEAS, could be used as precursors for the formation of androgens and estrogens (158). Support for this concept was obtained from the finding that relatively high STS activity was detected in ovarian follicles, stroma, and corpus luteum, which were capable of utilizing DHEAS as a substrate for the production of DHEA, androstenedione, and testosterone (159). DHEAS is present in high concentrations in follicular fluid in close proximity to the ovarian cells involved in steroidogenesis (160). Using human granulosa cells obtained from women undergoing treatment for in vitro fertility, significant conversion of DHEAS to DHEA was detected, confirming the presence of STS activity in these cells (32). Such conversion was effectively inhibited by the STS inhibitor EMATE. Addition of DHEAS to cultured granulosa cells stimulated estrogen production in a dose-dependent manner, demonstrating that granulosa cells can utilize DHEAS as a substrate for estrogen production. STS is also expressed in human fallopian tubes, which are involved in gamete transport and fertilization (22). Expression of STS was higher in fallopian tubes obtained from the early luteal phase than from the follicular phase of the menstrual cycle.

In addition to the role that steroid sulfates may have in breast cancer development, it is also likely that they may support the growth of hormone-dependent tumors in the reproductive tract of women. STS activity has been detected in normal and hyperplastic endometrial tissues (161). It has been suggested that uterine STS activity may have an important role in regulating the uterotropic activity of E2S (162). In a comparison of STS activities in malignant and normal endometrial tissues, activity was found to be 12-fold higher in malignant endometrial tissue (87). Sulfotransferase activity was also measured in this study and found to be significantly lower than STS activity, with no difference being detected between normal and malignant tissues. STS activity has also been detected in cultured cells derived from carcinomas of the ovary and vagina (163, 164). Using an immunohistochemical technique, positive STS expression was detected in 70% of ovarian clear cell adenocarcinoma tissue samples (19). Evidence showing that STS activity is present in hormone-sensitive tissues from the reproductive tract of women suggests that this enzyme may have an important role in regulating estrogen production in these tissues. With the development of potent STS inhibitors it will be possible to explore their therapeutic potential for the treatment of malignancies in the female reproductive tract.

2. Male, including the prostate gland.
STS is present in the testes of mammals, and it is likely that hydrolysis of steroid sulfates contributes to overall androgen production in this gland (165). An important role for STS has been postulated in the biochemical process of sperm maturation and capacitation (166). High levels of radiolabeled cholesterol sulfate are taken up by spermatozoa, and this was localized mainly within the plasma membrane of the acrosome region. It is thought that the cholesterol sulfate may act as a stabilizing factor that is associated with sperm membranes during transit or storage, inhibiting the release of acrosomal enzymes while sperm remain in the male reproductive tract. STS is present in the female reproductive tract, and hydrolysis of cholesterol sulfate by the STS may allow the release of acrosomal enzymes that facilitate penetration of the ovum by spermatozoa.

In males, the prostate gland is likely to be the major peripheral site where STS activity makes an important contribution to the production of biologically active androgens. It has been known for many years that men who have been castrated as part of their treatment for prostate cancer can have a further period of remission after adrenalectomy (167). The reason for this is thought to be due to the production by the adrenal cortex of weak androgens, such as DHEAS, that can be converted to testosterone and dihydrotestosterone in prostatic tissues (168). More recently, the combination of castration or LHRH agonist with an antiandrogen has been shown to result in an improved therapeutic response in subjects with prostate cancer (169). Whereas castration/LHRH agonist treatment removes the testicular source of androgen, the use of an antiandrogen is thought to block the action of androgen derived from the adrenal cortex.

STS activity has been detected in prostatic tissue (170). In studies in which the epithelial and stromal components of the prostate were separated, the highest STS activity was found to reside in the epithelial compartment (171, 172). LNCaP cells, which are derived from prostatic cancer, also possess STS activity, although at a somewhat lower level than that found in breast cancer cells (173). DHEAS was efficiently converted to DHEA in LNCaP cells, and hydrolysis of this steroid sulfate was almost completely blocked by the STS inhibitor, EMATE. The nonsteroidal inhibitor (p-O-sulfamoyl)-tetradecanoyl tyramine also inhibited the hydrolysis of DHEAS by these cells but was considerably less potent than EMATE. In view of the evidence supporting a role for STS in transforming weak androgens into biologically active androgen in the prostate, STS inhibitors could have considerable therapeutic potential for the treatment of prostate cancer.

F. STS activity in osteoblast cells
The reduction in ovarian estrogen production that occurs at the menopause has been implicated as an important factor in the development of osteoporosis. Because studies have generally failed to detect any consistent reduction in plasma estrogen concentrations in women with osteoporosis compared with women of similar age without fractures, the possibility of in situ estrogen synthesis by bone cells was considered (174). In three human osteoblast cell lines, HOS, MG 63, and U2 OS, the principal enzyme activities for estrogen synthesis, i.e., aromatase, 17ßHSD type 1, and STS, were all detected (29). STS activity in the MG 63 osteoblasts was 1000-fold higher than aromatase activity, suggesting that the local formation of E1 from E1S could be an important source of estrogen for regulating bone formation. In similar investigations, HOS and MG 63 osteoblasts were shown to express STS mRNA and to be capable of utilizing both E1S and DHEAS as substrates for STS activity (30).

In view of the potential importance of local formation of estrogens by osteoblasts for the maintenance of bone structure, it is possible that inhibition of STS could result in an increased rate of bone loss in treated subjects. However, studies with tibolone, which is used for hormone replacement therapy, have suggested that tibolone or its metabolites may have tissue-specific inhibitory effects on STS activity (175). After ingestion, tibolone is rapidly converted to metabolites that exert estrogenic effects or have progestogenic/androgenic properties (176). Although tibolone or its metabolites have estrogenic effects on bone and the central nervous system, no estrogenic stimulation of breast tissue occurs (177). In an attempt to find an explanation for these important effects, the ability of tibolone, its metabolites, or EMATE to inhibit STS activity in breast cancer cells, endometrial cells, or osteoblast cells was compared (175). All the compounds tested inhibited STS activity strongly in breast cancer cells and moderately in endometrial cells. In contrast, no significant inhibition of STS activity was detected in osteoblast cells. This study therefore raises the intriguing possibility that different tissues may express different isoforms of STS or may be subjected to different modes of regulation. Because STS inhibitors have now entered clinical trials, it will be important to confirm that compounds such as EMATE can act to inhibit STS activity in a tissue-specific manner.

G. STS activity in leukocytes and thrombocytes
In addition to the widespread distribution of STS in body tissues, the enzyme is also found in peripheral blood leukocytes (PBLs) and thrombocytes (26, 178). PBLs are capable of metabolizing steroid sulfates, and STS activity measurements, using these cells, have been used for the detection of the STS deficiency related to X-LI (179). Using [³H]E1S as a substrate, STS activity in PBLs from women in the follicular phase of their menstrual cycles was found to be almost twice as high as in cells collected from luteal phase subjects (26). This finding suggests that the high levels of progesterone present during the luteal phase may be involved in regulating STS activity. STS activity in PBLs obtained from men is lower than that in cells from female subjects (26, 180). Because many breast tumors are infiltrated by macrophages and lymphocytes (181), it is possible that the STS activity of these cells may make an important contribution to estrogen synthesis within tumors.

The presence of STS activity in a readily available tissue, such as PBLs, suggested that these cells could be used to provide a relatively simple method to monitor the extent and duration of STS inhibition when these drugs became available. This contrasts with the complex double-isotope infusion technique that is currently used to monitor aromatase inhibition in postmenopausal women (182). The discovery of the first potent STS inhibitor, EMATE, led to a preclinical study to evaluate the use of measuring STS activity in PBLs to determine the effectiveness of this inhibitor (183). Two hours after the oral administration of EMATE to rats, the extent of STS inhibition was similar in PBLs and liver. STS activity measurements in PBLs were also used to monitor inhibition of this enzyme in a preliminary male volunteer study in two subjects receiving 0.5 mg/kg EMATE (183). Assays of STS activity in PBLs from these subjects revealed that inhibition was almost complete by 4 h after dosing and was maintained for at least 1 wk. The ability to monitor the extent and duration of STS inhibition should be of considerable value when carrying out clinical trials to test the efficacy of this new form of therapy.

	VI. Tissue Availability of Steroid Sulfates

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
A central question with regard to the ability of steroid sulfates to exert physiological or pathological effects is whether they are taken up by cells, as such, or whether hydrolysis is a prerequisite for their entry into cells. It has generally been considered that although lipophilic unconjugated steroids are able to diffuse across cell membranes, polar hydrophilic steroid conjugates are unable to do so. Several studies have been carried out to investigate the uptake of steroid sulfates by cells and tissues using radiolabeled substrates, but these results have been difficult to interpret (103, 184).

In the last few years, convincing evidence has emerged for the existence of a super family of membrane transporter proteins (185). Some of these proteins appear to be involved in the specific uptake of organic anions, such as steroid sulfates, which have a negative charge and hydrophobic backbone. These transporters include the organic anion transporter and organic anion transporter polypeptide (OATP) proteins. Oatp1 was first identified in rats as a multispecific, sodium ion-independent transporter for a range of xenobiotics, bile acids, and conjugated metabolites (186). Subsequently, other structurally related homologs of Oatp1, Oatp2, and Oatp3 were isolated (187, 188). In humans OATP was originally cloned from a human liver-derived cDNA library as a homolog of rat Oatp1 (189). A series of related homologs was subsequently identified in humans (OATP-B, OATP-C, OATP-D, and OATP-E), which were expressed at varying levels in a wide range of tissues (190). OATP-E was expressed in several different cancer cell lines, whereas OATP-D was not expressed in G1–101 breast cancer cells. Functional studies with human embryonic kidney (HEK)-293 cells transfected with the cDNAs for the different transporters revealed that whereas OATP-B, -C, -D, and -E all transported E1S, the highest activity was observed for OATP-B and OATP-C. Results from these studies show that OATPs are active transporters for E1S.

Using an immunohistochemical technique, OATP-B was recently found to be highly expressed in the human mammary gland (191). Because the main substrate for OATP-B is E1S, it was suggested that the major physiological function of this carrier in the mammary gland is the uptake of E1S. In the normal mammary gland, OATP-B expression was confined to myoepithelial cells. Because these cells have been found to possess STS activity (192), it was postulated that the myoepithelial cells may be responsible for supplying nonsulfated estrogen to the adjacent epithelial cells. The major finding to emerge from this study was that OATP-B is strongly expressed in the majority of epithelial cells in invasive ductal carcinomas. In related in vitro studies, uptake of E1S and DHEAS by OATP-B was found to be stimulated by prostaglandin A1 and prostaglandin A2, suggesting that the uptake of steroid sulfates could be regulated locally at the plasma membrane.

Because the plasma concentrations of E1S are much higher than those of unconjugated E1 or E2, the finding of a specific transporter for steroid sulfates in malignant breast tissues is of considerable importance. STS activity and expression, as previously discussed, are elevated in breast tumors. Thus, all the elements are in place in breast tumors to ensure the efficient uptake of E1S from the circulation and its rapid hydrolysis to a biologically active estrogen. This combination of an effective transporter for E1S and high STS activity offers a likely explanation for the high concentrations of E1 and E2 that are found in breast tumors.

	VII. STS Inhibitors

Top Abstract I. Introduction II. Molecular Biology of... III. Localization of STS IV. Regulation of STS... V. Biological Roles of... VI. Tissue Availability of... VII. STS Inhibitors VIII. Active Pharmacophore... IX. In Vivo Activity... X. Dual-Function Inhibitors XI. Mechanism of Steroid... XII. Future Perspectives References

 
In view of the important roles of STS in physiological and pathological conditions, considerable research has been carried out to develop potent inhibitors of this enzyme (78, 79, 80, 81, 193, 194, 195).

A. Alternative substrates
This type of compound, which contains at least one sulfate group in the structure, is designed to compete with E1S for binding to the STS enzyme active site and, as a consequence, impede the hydrolysis of the natural substrate to E1. These inhibitors, in principle, are alternative substrates for STS, the sulfate group(s) of which are expected to be hydrolyzed by the enzyme. The very first example of such a class of STS inhibitor was a series of 2-(hydroxyphenyl) indole sulfates, one of which (Fig. 4, compound 1) showed an IC₅₀ value of 80 µM (196). Several synthetic and naturally occurring steroids were also investigated for STS-inhibitory activity, of which 5-androstene-3ß,17ß-diol-3-sulfate (Fig. 4, compound 2) was found to be the most potent [inhibition constant (K_i) = 2.0 µM (197)]. Flavonoids daidzein 4'-O-sulfate (Fig. 4, compound 4) and daidzein 4',7-di-O-sulfate (Fig. 4, compound 5) were synthesized and found to inhibit STS competitively with K_i values of 5.9 and 1 µM, respectively (198). However, inhibitors such as compounds 2, 4, and 5 could potentially be problematic because their corresponding metabolites, androstenediol (Fig. 4, compound 3) and daidzein (Fig. 4, compound 6), are known estrogens, which renders them of little value clinically for the treatment of hormone-dependent breast cancer.

View larger version (9K): [in this window] [in a new window]   FIG. 4. Structures of alternative substrates acting as inhibitors of STS.

View larger version (28K): [in this window] [in a new window]   FIG. 5. Examples of reversible STS inhibitors.

Derivatives of 17-benzylestradiol (Fig. 5, compound 12) bearing a 4'-t-butyl (Fig. 5, compound 13), 3'-bromo (Fig. 5, compound 14), or 4'-benzyloxy (Fig. 5, compound 15) substituent were among the most potent reversible inhibitors reported to date, showing IC₅₀ values (JEG-3 cells) between 22 and 28 nM (213, 214). It was found that compound 13 was about 7-fold weaker than EMATE as an STS inhibitor when tested in a transfected HEK-293 cell preparation (214). A series of 17-alkan- or alkynamide derivatives of E2 were prepared, and the propanamide 16 (Fig. 5) gave an IC₅₀ of 80 nM in JEG-3 cells (215). The relatively high potency against STS observed for compound 16 is evidence of exploitation by the hydrophobic substituent of the hydrophobic binding area(s) that have been postulated to be in the vicinity of the D ring of EMATE.

In an attempt to overcome the unwanted estrogenicity of some 17-substituted derivatives of EMATE (Fig. 6, compounds 36 and 37) (see Section VII.C), several sulfamates of C19 (androstene) or C21 (pregnene) derivative were prepared (Fig. 5, compounds 17–19) (216). 17-t-Butylbenzyl-5-androsten-17ß-ol (compound 19) was the best reversible inhibitor (IC₅₀ = 46 nM) in a homogenate preparation of HEK-293 cells and showed no estrogenic or androgenic activities in vitro (216).