Steroid Sulphotransferase Research Articles

Abstract 808: Endocrine resistance: The influence of stromal-epithelial interaction on intracrine pathways of oestrogen synthesis

Abstract Resistance to endocrine therapy, both de novo and acquired, poses a significant clinical challenge. Two primary pathways, the aromatase and steroid sulphatase (STS) pathways, exist for the production of estrogenic steroids from androstenedione and DHEAS, respectively. Numerous studies have demonstrated an upregulation of these pathways in breast tumour tissue compared to normal breast tissue. Therapies targeting oestrogenic pathways form the mainstay of the pharmacological approach to breast cancer treatment. The SERM, Tamoxifen, and aromatase inhibitors (AI) such as Letrozole, have shown substantial clinical benefit in hormone-dependent breast cancer patients. Additionally, to date one STS inhibitor has entered clinical trials. Despite this success, a proportion of hormone-receptor positive patients do not respond to hormone therapy with 40% of patients who initially show a response to Tamoxifen relapsing. As little is known about the influence of the tumour microenvironment, particularly stromal-fibroblasts, on these pathways in the development of endocrine resistance, we have examined the influence of stromal-epithelial interactions on the expression of enzymes of steroidogenesis in endocrine resistant cells. Wild type and tamoxifen-resistant MCF-7 breast cancer cells (MCF-7wt and MCF-7TamR, respectively) were indirectly co-cultured with primary fibroblasts derived from breast tumour and reduction mammoplasty samples, using 0.4μm 6-well inserts. Expression levels of 17α-hydroxysteroid dehydrogenase types 1 (17α-HSD1) and 2, (17α-HSD2), aromatase, oestrogen receptor ≤ (ERα) and β, progesterone receptor, steroid sulphatase (STS), and steroid sulphotransferase were assessed basally and in co-culture using RT-PCR. Significantly higher basal expression of STS (p<0.001), aromatase (p=0.012) and 17α-HSD2 (p=0.032) was seen in fibroblast versus epithelial lines. On co-culture a 2.5 - 5 fold up-regulation of aromatase expression was seen in MCF-7wt and MCF-7TamR (p=0.009, p=0.046 respectively). The expression of STS (p=0.150, p=0.05) and 17α-HSD1 (p=0.001, p=0.071) was also increased. Epithelial lines showed a significant down-regulation in ERα on co-culture. Both malignant and normal fibroblast lines demonstrated up-regulation of aromatase expression on co-culture (p=0.037). These data suggest that stromal-fibroblasts make a significant contribution to the tumour hormonal environment through both expression and modulation of the pathways associated with intracrine oestrogen production. The differential effect of cell-type combinations implicates these interactions in both oestrogen sensitivity and insensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 808. doi:1538-7445.AM2012-808

Tibolone and its delta-4, 7α-methyl norethisterone metabolite are reversible inhibitors of human aromatase

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3α- and 3β-hydroxy derivatives and the delta-4, 7α-methylnorethisterone (7α-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase Type 1 (17β-HSD1) activities but also stimulate steroid sulphotransferase and 17β-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7α-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7α-MeNET, but not the 3α- or 3β-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10 μM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7α-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17β-HSD1, tibolone and 7α-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.

Steroid sulphotransferase and 17beta-hydroxysteroid dehydrogenase activities in Ishikawa human endometrial adenocarcinoma cells.

The present studies concern sulphotransferase activities for estrogens and other steroids, and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities for estrogens in Ishikawa endometrial adenocarcinoma cells. When physiological concentrations of various estrogens (estrone, estradiol, estriol) are incubated, most of the transformation product is the respective sulphate. The sulphotransferase activity is very rapid, and 2 h after incubation 70-95% are converted to the sulphated form. Sulphates are found exclusively in the culture medium, which suggests that as soon as the sulphate is biosynthesized it is secreted to the medium. Comparative data using neutral steroids (dehydroepiandrosterone, testosterone, and pregnenolone) show that sulphotransferase activity for these compounds is very limited. In another series of studies, 17beta-HSD activity was explored for the interconversion estrone estradiol. At low concentrations (5 x 10(-9)-5 x 10(-8) M), when estradiol (E2) is incubated, most of the unconjugated material remains as E2 in the cellular compartment, but at high concentrations (5 x 10(-7)-5 x 10(-6) M) a great proportion (70-80%) of the E2 is converted to estrone (E1). On the other hand, after incubation of E1 at all concentrations most remained as unchanged E1. It is suggested that, in Ishikawa cells, at very low concentrations of E1 or E2, sulphotransferases are predominant, but when this enzyme is saturated 17beta-HSD activity is orientated to the oxidative form.

Steroid sulphotransferase activity in human hormone-independent MDA-MB-468 mammary cancer cells

The metabolism of various unconjugated oestrogens [oestrone (E 1), oestradiol (E 2) and oestriol (E 3)], in MDA-MB-468 human mammary cancer cells, a cell line characterised by the absence of oestrogen receptor has been investigated. 24 h after incubation of these cells with 5 × 10 −9 mol/l of tritiated E 1, E 2, or E 3, 62–90% of the total radioactivity was localised in the sulphate fraction. Analysis showed that 73–90% of the sulphate fraction corresponds with the oestrogen incubated. The formation of the oestrogen sulphates is rapid and maximum values are found after 3 h incubation. Intense sulphotransferase activity was also found for testosterone, pregnenolone and dehydroepiandrosterone. Thus these cells contained high oestrogen sulphotransferase activity, suggesting that the presence of high levels of oestrogen sulphates in breast cancer can be synthesised in the tumour itself. In addition, the control of this enzymatic activity could open new possibilities in the knowledge of oestrogen responses and of therapeutic applications in breast cancer.

Human liver steroid sulphotransferase sulphates bile acids

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.

Liver-specific expression of paracetamol sulphotransferase

Conjugation reactions constitute the principal routes of dcactivation and elimination of potentially toxic endoand xcnobiotics [ I ] . Conjugation with sulphate, catalysed by the family o f sulphotransferase (ST ) enzymes, is a key mechanism for metabolism of many endogenous compounds such as neurotransmitters. steroid hormones and bile salts; however, many phenolic and amine drugs and carcinogens are also metabolized by this route [2]. Heterogeneity of STs has been determined principally by purification of the different isoenzymes, and they are divided into subfamilies according to the chemical properties of their substrates, e.g. phenol STs (PSTs), steroid STs, bile salt STs, oestrone STs [ 31. Paracetamol is an extremely widely used analgesic/antipyretic agent, which is metabolized, in therapeutic dose, to its sulphate and glucuronide conjugates. However, in overdose it causes severe or fatal hepatic and/or renal necrosis. We have recently identified and purified a form of rat liver PST responsible for the sulphation of paracetamol (M. Coughtrie & S. Sharp, unpublished work).

Cytosolic phenol and steroid sulphotransferase activities are decreased in a sex-dependent manner in streptozotocin-induced diabetic rats

Cytosolic phenol and steroid sulphotransferase activities are decreased in a sex-dependent manner in streptozotocin-induced diabetic rats

Identification and partial purification of a unique phenolic steroid sulphotransferase in rat liver cytosol.

Phenolic steroid sulphotransferase activity for both oestradiol and oestrone was identified in male rat liver cytosol in the 30 000-40 000 Mr fractions on gel filtration when activity was assayed at pH 5.5 (pH optimum 5.5-6.0). Activity for oestradiol but not oestrone was found in the 60 000-70 000-Mr range when assayed at pH 8.0 (pH optimum biphasic, 5.5-6.0 and 7.0-8.0). Km for oestradiol (1.3 microM) was lower than published values for hydroxysteroid sulphotransferases (15-35 microM) and previously reported oestradiol sulphotransferases (71-85 microM). At above 2 microM-oestradiol phenolic sulphotransferase activity exhibited substrate inhibition. The phenolic steroid sulphotransferase activity was found to be distinct in chromatofocusing from organic-anion-binding and bile acid-binding proteins previously identified in this Mr range. Further purification on hydroxyapatite yielded a 44-fold enriched fraction that contained two monomeric bands, Mr 32 500 and 29 500.

Sulphoconjugation of steroids in porcine liver: Partial purification of the cytosolic sulphotransferases for pregnenolone and 5α-androst-16-en-3β-ol

Steroid sulphotransferase activities for 5α-androst-16-en-3β-ol and pregnenolone in porcine liver cytosol have been assayed using 3′-phosphoadenosine-5′-phospho[ 35S]sulphate as sulphate donor. 5α-Androst-16-en-3β-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37°C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KCl. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37°C, respectively.

Studies on a 3β3-hydroxysteroid sulphotransferase from rat liver

A steroid sulphotransferase (EC 2.8.2.2) was partially purified from female rat liver. The enzyme was active towards the substrates, dehydroepiandrosterone, epiandrosterone and pregnenolone but was inactive towards oestrogens, cholesterol and ergocalciferol. A pH optimum of 5.0 was recorded but the enzyme was unstable at low pH. The enzyme was stimulated slightly by the addition of reducing agents and inhibited by p- chloromercuribenzoate and HgCl 2. Crude enzyme activity was markedly stimulated by divalent cations but this effect was not observed with purified enzyme. A K m of 13 μM was calculated for the donor substrate 3′-phosphoadenylyl sulphate and the acceptor substrate, dehydroepiandrosterone had a K m value of 6 μM. The enzyme appeared to be highly susceptible to product inhibition by adenosine 3′,5′diphosphate.

Reinigung und charakterisierung einer 3′-phosphoadenylylsulfat:steroid-sulfotransferase aus der leber des menschen

A 3′-phosphoadenylylsulphate:steroid sulphotransferase is localised in the 150 000 × g supernatant of normal human liver. The enzyme was purified 22-fold by (NH 4) 2SO 4 precipitation, by gel filtration of the precipitate through Sephadex G-200 and by chromatography on DEAE-cellulose. After incubation of the enzyme with dehydroepiandrosterone as substrate, the reaction product isolated was identified as dehydroepiandrosterone sulphate by paper chromatography, various micro-chemical reactions and crystallization with authentic carrier to constant specific activity. The sulphotransferase showed a pH optimum at 7.4 in Tris-HCl buffer. The Michaelis-Menten constants were found to be 6.7·10 −6 M for dehydroepiandrosterone and 4.0·10 −6 M for 3′-phosphoadenylylsulphate. The enzyme lost its activity within 6 days when stored at 4°; however, at −20°, the enzyme was stable for at least 4 months. The steroid sulphotransferase showed a temperature optimum at 50° and an activation energy of 22.2 kcal/mole, within the range of 37–50°. By gel filtration, using Sephadex G-200, the molecular weight of the sulphotransferase was found to be approximately 50 000. The activity of the purified enzyme preparation was not influenced by the addition of cystein or EDTA. In relatively high concentration, SH-blocking agents had an inhibitory effect. The addition of Co 2+, Ni 2+, Ca 2+ and Mn 2+ activated the enzyme, whereas Cu 2+, Zn 2+, Fe 2+ and Hg 2+ inhibited the sulphotransferase. The steroid sulphotransferase from human liver conjugates not only dehydro-epiandrosterone (100%), but also (although to a lower extent) 3α-hydroxy-5β-androstan-17-one (45%), oestriol (31%), oestradiol-17β (15%) and oestrone (8%). Testosterone and cholesterol were not conjugated by the enzyme.

Enzymic synthesis of steroid sulphates: IV. The nature of the two forms of estrogen sulphotransferase of bovine adrenals

1. 1. The nature of the two forms (A and B) of estrogen sulphotransferase (3′- phospoadenylylsulphate: estrone sulphotransferase, EC 2.8.2.4) isolated by DEAE-cellulose chromatography, has been investigated. In contrast to the A form, the B form gave non-linear double-reciprocal kinetic plots. Similar plots were obtained when cysteine was added to the A form. 2. 2. Form B was shown to be converted to Form A on standing. 3. 3. Only the B form was obtained when the enzyme was isolated in the presence of mercaptoethanol. 4. 4. Form B was shown to be related to A as trimer (or perhaps tetramer) to monomer, by thin-layer chromatography on Sephadex G-200. The molecular weight of the monomer was estimated to be about 67 000 by this method. 5. 5. The ability to associate to the trimer is suggested to be related to the possession of a conformation dependent on the maintenance of a SH group, or groups, in the reduced state. Non-linear kinetic curves are explained by interaction of binding sites on the associated protein. 6. 6. Allosteric properties exhibited by the fully-associated enzyme are discussed in terms of control mechanisms which may operate at the level of steroid sulphotransferase in endocrine organs. 7. 7. Both forms showed three to four closely migrating protein bands on acrylamide gel electrophoresis which persisted throughout the purification procedures. Evidence was presented that these bands represented individual isoenzymes. 8. 8. The amino acid composition of the highly purified enzyme has been determined.