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Differential effects of progestins on breast tissue enzymes
Maturitas 46S1 (2003) S45–S54
Differential effects of progestins on breast tissue enzymes J.R. Pasqualini∗ Hormones and Cancer Research Unit, Institut de Puériculture, 26 Blvd. Brune, Paris 75014, France
Abstract There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E2 ) from circulating precursors. Two principal pathways are implicated in the final steps of E2 formation in breast cancer tissue: the ‘aromatase pathway’ that transforms androgens into estrogens and the ‘sulfatase pathway’ that converts estrone sulfate (E1 S) into estrone (E1 ) via estrone sulfatase. The final step is the conversion of weak E1 to potent biologically active E2 via reductive 17-hydroxysteroid dehydrogenase type 1 activity. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are present in breast cancer tissues. One of the possible means of blocking E2 effects in breast cancer is to use anti-estrogens, which act by binding to the estrogen receptor (ER). Another option is to block E2 using anti-enzymes (anti-sulfatase, anti-aromatase, or anti-17-hydroxysteroid dehydrogenase (17-HSD). Various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, 17-deacetyl norgestimate, dydrogesterone and its 20-dihydro derivative), as well as tibolone and its metabolites, have been shown to inhibit estrone sulfatase and 17-hydroxysteroid dehydrogenase. Some progestins and tibolone can also stimulate sulfotransferase activity. These various progestins may therefore provide a new option for the treatment of breast cancer. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: 17-Hydroxysteroid dehydrogenase; Progestin; Breast cancer
1. Introduction The “intracrine concept”, where a hormone has a biological response in the same organ in which it is produced, is entirely applicable to breast carcinoma tissue for two main reasons: (1) breast cancer tissue accumulates extensive quantities of estrogens (unconjugates and sulfoconjugates), particularly in postmenopausal patients; (2) the enzyme systems for the bioformation and metabolic transformation of estrogens are present in high levels in this tissue. It is clear that the biological responses to steroid hor∗
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mones in their target tissue are subject to modulation by a variety of enzymes. These enzymes may control the access of steroids to their receptors by converting them into forms with a higher affinity for the receptor or by inactivating the corresponding steroid. One of the possible ways of blocking the estradiol (E2 ) effect in breast cancer is via anti-estrogens. Another way to block E2 is by using anti-enzymes (anti-sulfatase, anti-aromatase, or anti-17-hydroxysteroid dehydrogenase (17-HSD) (type 1 against the enzymes that are involved in E2 biosynthesis in breast cancer tissues)). At present, anti-aromatases are extensively used in breast cancer treatment with positive benefits. However, estrone sulfatase is quantitatively the most important pathway in E2 bioformation in
0378-5122/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2003.09.018
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breast cancer tissue. In this review, we summarise the effect of various progestins on estrone sulfatase, 17-hydroxysteroid dehydrogenase and estrone sulfotransferase in human breast cancer tissue.
2. Estrone sulfatase and its control in breast cancer 2.1. The importance of estrone sulfatase in breast cancer E2 is one of the most important factors in the growth and evolution of hormone-dependent breast tumors and it is now well accepted that breast tumors have the capacity to locally synthesize active E2 . Two metabolic pathways are implicated in this intra-tumoral biosynthesis: the “aromatase pathway” that converts adrenal androgen precursors (e.g. androstenedione) to estrone (E1 ) by aromatisation, followed by reduction of estrone to E2 via 17-hydroxysteroid dehydrogenase (17-HSD) type 1; and the “sulfatase pathway” that transforms the precursor estrone sulfate (E1 S) to E1 via sulfatase in adipose tissues, followed by 17-HSD type 1 reductive activity to E2 . Estrogen sulfates and sulfatases are important in breast cancer due to the following reasons: 1. Estrone sulfate is the most abundant circulating estrogen in the plasma of postmenopausal women. The levels of this conjugate are 2–10 times higher than those of unconjugated E1 and E2 [1–3]. 2. As estrogen sulfates do not bind to estrogen receptors (ER), estrogen-3-sulfates must be hydrolyzed by sulfatases to elicit a biological response. E1 , after conversion to E2 , increases the amount of progesterone receptors (PR) and pS2 protein, as well as estrogen-inducible protein cathepsin D [4–8] in hormone-dependent breast cancer cells (MCF-7), whereas estrogen-17-sulfates, which are not hydrolyzed by sulfatases, do not cause any biological responses [6,7]. 3. The incidence of breast cancer is higher after the menopause when the ovaries have ceased to be functional and the levels of E2 and estrone sulfate are 7–11 times higher in breast tumour tissue than in plasma [3].
4. The concentration of estrone sulfate in the tumor is higher in postmenopausal than in premenopausal breast cancer patients [3]. 5. The tissue concentration of estrone sulfate is higher in tumoral than in normal breast tissue [9]. 6. Sulfatase activity is very intense in malignant and benign breast tumors compared with normal breast tissue [9–12]. 7. Quantitative measurements indicate that levels of estrone sulfatase are 40–500 times higher than those of aromatase in breast tumour tissue [3]. 2.2. Control of estrone sulfatase in breast cancer Estrone sulfatase activity is high in human hormone-dependent breast cancer cells (MCF-7, T-47D). In contrast, hormone-independent breast cancer cells (MDA-MB-231, MDA-MB-468) show very low sulfatase activity in intact cells, but the activity is restored when the cells are homogenized [13,14]. The sulfatase mRNA is present in both the hormone-dependent and hormone-independent breast cancer cells, and the expression of this mRNA correlates with sulfatase activity [15]. The data clearly indicate that sulfatase is present in hormone-independent cells, but that it does not function in complete cells. Why, in spite of the existence of the enzyme, is very little estrone sulfate hydrolyzed within these intact cells? The answer to this question is not currently clear, but suggestions include the presence of repressive factor(s) or sequestration into an inactive form for this kind of cell. More information is needed to elucidate this mechanism. As estrone sulfatase is the other important means of producing biologically active estrogens, blockade of the sulfatase pathway constitutes a promising alternative for reducing the level of E2 . In recent years, the potential inhibitory effects of a wide range of compounds, including anti-estrogens, progestins, tibolone and its metabolites, as well as steroidal and non-steroidal compounds, have been explored. 2.2.1. Inhibition by anti-estrogens The anti-estrogen tamoxifen and its more potent metabolite, 4-hydroxytamoxifen, as well as ICI 164,384, have been reported to inhibit sulfatase activity, probably by a non-competitive mechanism [16–19]. Chu et al. [20] found that, in the rat liver,
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Fig. 1. Comparative effects of various progestins on inhibition of estrone sulfate (E1 S) conversion to estradiol (E2 ) in hormone-dependent T-47D human breast cancer cells. The values represent mean ± S.E.M. of duplicate determinations of three–seven independent experiments. R-5020: promegestone; Nom. Ac.: nomegestrol acetate; Medrog.: medrogestone; Noreth.: norethisterone. ∗ P ≤ 0.05 vs. control value; ∗∗ P ≤ 0.01 vs. control value. From Chetrite and Pasqualini [59].
(E)- and (Z)-4-hydroxytamoxifen sulfamates are also sulfatase inhibitors, with Ki values of 35.9 and >500 M, respectively. 2.2.2. Inhibition by progestins Incubation of physiological concentrations (5 × 10−9 M) of estrone sulfate with MCF-7 or T-47D human breast cancer cell lines showed that various progestins (nomegestrol acetate, promegestone, progesterone, medrogestone (Prothil® ), 17-deacetyl norgestimate can provoke significant inhibition of estrone sulfatase activity [13,21–24]. Fig. 1 shows the relative percentage of sulfatase inhibition by theseprogestins. Recently, it was demonstrated that dydrogesterone (Duphaston® ) and its 20-dihydro derivatives are potent inhibitors of estrone sulfatase in MCF-7 and T-47D breast cancer cells [25]. This is indicated in Fig. 2. 2.2.3. Inhibition by tibolone and its metabolites Tibolone (Org OD14; active substance of Livial® (2.5 mg)) is a 19-nortestosterone derivative with tissue-specific estrogenic, progestogenic and androgenic properties. It is used as monotherapy for the treatment of climacteric symptoms and the prevention of osteoporosis, without stimulating the endometrium. Tibolone, and its more active metabolites Org 4094, Org 30,126 (3␣- and 3-hydroxy derivatives) and
Org OM38 (the 4-en isomer), are potent sulfatase inhibitors at low concentrations (5 × 10−7 M) in MCF-7 and T-47D hormone-dependent breast cancer cells [26].
Fig. 2. Effects of dydrogesterone (DYD) and 20-dihydro derivate (DHD) on the conversion of estrone sulfate (E1 S) to estradiol (E2 ) in the hormone-dependent MCF-7 human breast cancer cell line. Cells were incubated for 24 h at 37 ◦ C with a physiological concentration (5 × 10−9 mol/l) of [3 H]-E1 S alone (control: non-treated cells) or in the presence of DYD or DHD at the range of concentrations from 5 × 10−9 mol/l to 5 × 10−5 mol/l. Qualitative and quantitative analyses of E2 in the cell compartment were performed by thin-layer chromatography and liquid scintillation counting. Results are expressed in fmol of E2 formed/mg DNA from E1 S. The data are the mean ± S.E.M. of duplicate determinations of three independent experiments. ∗ P = 0.05 vs. control values (non-treated cells).
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2.2.4. Inhibition by steroidal compounds Estrone-3-O-sulfamate (EMATE) is a potent synthesized sulfatase inhibitor [27]; at a concentration of 10−7 M, estrone sulfatase activity in MCF-7 cells is 99% inhibited [28,29]. Unfortunately, the potent estrogenic activity of this compound precludes its use in clinical applications [30,31]. Estrone phosphate and DHEA-phosphate are also potent inhibitors of estrogen sulfatase activity [32]. In other studies, Boivin et al. [33] and Poirier and Boivin [34] attempted to develop sulfatase inhibitors without residual estrogenic activity by synthesizing a series of E2 derivatives bearing an alkyl, a phenyl, a benzyl substituted (or not), or an alkyan amide side chain at position 17␣. These studies showed that sulfatase inhibitors act by a reversible mechanism and that the hydrophobic group at the 17␣ position increases the inhibitory activity, whilst stearic factors contribute to the opposite effect. The most potent inhibitor is a 17␣-benzyl substituted E2 derivative with an IC50 value of 22 nM. When these 17␣-substituents were added to the 3-O-sulfamate estradiol structure, the combined inhibitory effect was more potent, with an IC50 value of 0.15 nM [35]. 2.2.5. Inhibition by non-steroidal compounds An interesting new family of compounds has been synthesized with a tricyclic coumarin sulfamate structure [36–40]. These non-steroidal sulfatase inhibitors are active in vitro and in vivo, are non-estrogenic and possess, in vitro, an IC50 value of approximately 1 nM. However, the most potent inhibitor in vivo does not correspond to the best compound in vitro. 2.2.6. Inhibition of estrone sulfatase activity by estradiol (E2 ) Recent studies have demonstrated a paradoxical effect of E2 in MCF-7 and T-47D breast cancer cells in that it can block its own bioformation by inhibiting, in a dose-dependent manner, the conversion of estrone sulfate to E2 at concentrations of 5 × 10−10 M to 5 × 10−5 M [41] (Fig. 3). E2 is a potent inhibitor of estrone sulfatase activity, with IC50 values of 1.84 × 10−9 M and 8.77 × 10−10 M in T-47D and MCF-7 cells, respectively.
Fig. 3. Effect of estradiol (E2 ) on the conversion of estrone sulfate (E1 S) to E2 in MCF-7 human breast cancer cells. The percentage of inhibition was obtained by calculating the ratio [(control − test)/control] × 100. The values represent mean ± S.E.M. of duplicate determinations of five independent experiments. ∗ P ≤ 0.05 vs. control value; ∗∗ P ≤ 0.005 vs. control value. From Pasqualini and Chetrite [41].
3. 17-Hydroxysteroid dehydrogenase in the breast 17-Hydroxysteroid dehydrogenase (17-HSD), a widely distributed enzyme in mammalian tissues, is implicated in the inter-conversion of the inactive 17-keto to the active 17-hydroxy form of sex steroid hormones (estrogens and androgens). However, some types of 17-HSD may metabolize other substrates such as bile acids, alcohols, fatty acids and retinols. 17-HSD belongs to a superfamily of enzymes in which 11 different isoforms have been recognised to date. In normal breast tissue, oxidative 17-HSD activity (E2 to E1 ) was found to be the preferential direction and this activity was more intense during the secretory phase of the menstrual cycle [42]; 17-HSD type 1 and 2 mRNA are both expressed in glandular epithelium. In HME human mammary epithelial cell lines, mRNA for 17-HSD types 1, 2, and 4 was detected, but only oxidative 17-HSD activity was present; it
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was suggested that this activity is due to 17-HSD type 2 [43]. In breast tumours, in vivo and in vitro studies show that the preferential conversion is the reduction of E1 to E2 . 17-HSD type 1 is found in the cytoplasm of malignant epithelial cells in breast tumours [44]. However, the direction of enzymatic activity (oxidative or reductive) in breast cancer is also greatly dependent on the local, metabolic or experimental conditions, including: the nature and concentration of the cofactors (e.g. NADPH or NADP) and substrate, pH, and the subcellular localisation of enzymes. In vitro studies using human tumour homogenates indicated that the predominant 17-HSD activity was oxidative rather than reductive [45]. However, in vivo studies following isotopic infusion of estrogens in postmenopausal breast cancer patients have shown that the reductive direction is greater than the oxidative [44]. In hormone-dependent breast cancer cell lines (MCF-7, T-47D, R-27, ZR-75-1), 17-HSD type 1 was found to be the predominant reductive isoform, but types 2 and 4 isoforms with oxidative activities (formation of E1 ) were also detected [44,46–48]. 3.1. Control of 17β-HSD in the breast In epithelial cells from normal breast, the progestin promegestone (R-5020) was shown to increase 17-HSD activity in the oxidative (E2 to E1 ) direc-
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tion; this stimulatory effect of progestins depends on preliminary sensitization by estrogens [49,50]. Progestins can induce 17-HSD type 1 activity, with an increase in both the 1.3 kb mRNA species and the enzyme protein, in hormone-dependent T-47D breast cancer cells [44,48]. Coldham and James [51] found that the progestin medroxyprogesterone acetate (MPA) stimulated the reductive (E1 to E2 ) activity of MCF-7 cells when phenol red was excluded from the tissue culture media. The authors suggested that this could be the way in which progestins increase cell proliferation in vivo. On the other hand, Couture et al. [46] observed that when hormone-dependent ZR-75-1 breast cancer cells were incubated with MPA, the oxidative (E2 to E1 ) direction was predominant; this effect seems to implicate the androgen receptor. Other progestins, such as progesterone, levonorgestrel and norethisterone, increased both the oxidative and reductive 17-HSD activity in MCF-7 cells [52], whereas promegestone (R-5020) had no significant effect on the reductive activity of 17-HSD [53] but increased the oxidative (E2 to E1 ) activity in T-47D cells [54]. Nomegestrol acetate had an inhibitory effect on the 17-HSD enzyme in T-47D cells (35 and 81% inhibition at 5 × 10−7 M and 5 × 10−6 M, respectively), but no significant effect was found in MCF-7 cells, except at 5 × 10−5 M [22]. Medrogestone (Prothil® ), a synthetic pregnane derivative of progesterone, significantly decreased the reductive 17-HSD type 1 activity in MCF-7 and T-47D breast cancer cells. The
Fig. 4. Comparative effects of various progestins on inhibition of the conversion of estrone (E1 ) to estradiol (E2 ) in hormone-dependent T-47D human breast cancer cells. The values represent mean ± S.E.M. of duplicate determinations of three–six independent experiments. R-5020: promegestone; Danaz.: danazol; Medrog.: medrogestone; Nom. Ac.: nomegestrol acetate. ∗ P ≤ 0.05 vs. control value. From Chetrite and Pasqualini [59].
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inhibitory effect was concentration-dependent and more intense, even at low concentrations, in the T-47D cell line than in the MCF-7 cells; the IC50 values, which correspond to 50% inhibition of the conversion of E1 to E2 , were 0.45 and 17.36 M, respectively [55]. Comparative effects of various progestins: promegestone, danazol, medrogestone, nomegestrol acetate, on the inhibitory effect of 17-HSD in T-47D cells are indicated in Fig. 4. Recently, it was shown that dydrogesterone (Duphaston® ) and its 20-dihydro derivatives can also inhibit the conversion of E1 to E2 in the T-47D breast cancer cells (Fig. 5). Fig. 5. Effects of dydrogesterone (DYD) and its 20-dihydro derivate (DHD) on the conversion of estrone (E1 ) to estradiol (E2 ) in the hormone-dependent T-47D human breast cancer cell line. Cells were incubated for 24 h at 37 ◦ C with a physiological concentration (5 × 10−9 mol/l) of [3 H]-E1 alone (control: non-treated cells) or in the presence of DYD or DHD at the range of concentrations from 5 × 10−9 mol/l to 5 × 10−5 mol/l. Qualitative and quantitative analyses of E2 in the cell compartment were performed by thin-layer chromatography and liquid scintillation counting. Results are expressed in fmol of E2 formed/mg DNA from E1 . The data are the mean ± S.E.M. of duplicate determinations of three independent experiments. ∗ P = 0.05 vs. control values (non-treated cells).
4. Sulfotransferase and its control in breast cancer As sulfoconjugates are not biologically active, control of their formation in breast cells represents an important mechanism by which to modulate the biological action of E2 in this tissue. Comparative studies on the formation of estrogen sulfates after incubation of E1 with hormone-dependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231) breast cancer
Fig. 6. Effects of medrogestone (Prothil® ) on the conversion of estrone (E1 ) to estrogen sulfates in hormone-dependent MCF-7 and T-47D human breast cancer cells. Results (pmol of ES formed/mg DNA) are expressed in percent of control values considered as 100%. The values represent mean ± S.E.M. of duplicate determinations of three independent experiments. ∗ P ≤ 0.05 vs. control value (non-treated cells). ∗∗ P ≤ 0.01 vs. control value (non-treated cells). From Chetrite et al. [23].
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cells show significantly higher sulfotransferase levels in the former [55]. 4.1. Effect of medrogestone and other progestins Medrogestone is a synthetic pregnane derivative used in the treatment of pathological deficiency of natural progesterone. This compound has secretory activity in the estrogen-primed uterus, is thermogenic and acts as an anti-estrogen and antigonadotropin. In MCF-7 and T-47D breast cancer cells, medrogestone had a bi-phasic effect on sulfotransferase activity: at a low concentration (5 × 10−8 mol/l) it stimulated the formation of estrogen sulfates in both cells lines, whereas at a high concentration (5 × 10−5 mol/l) sulfotransferase activity was not modified in MCF-7 cells or inhibited in T-47D cells [23] (Fig. 6).
Fig. 8. Effects of estradiol sulfotransferase (EST) activity on the proliferation of breast cancer cells. In normal breast cells, as a consequence of EST activity, proliferation is inhibited because estradiol sulfate (E2 S) is biologically inactive. In contrast, in breast cancer cells, hEST activity is very low or non-existent as E2 S is not formed and E2 can stimulate proliferation.
Hypothetical effect of Medrogestone on Sulfotransferase (hEST) and Proliferation in MCF-7 cells I) MEDROGESTONE stimulates hEST
5. Possible correlation of sulfotransferase activity and proliferation of breast cancer cells
II) hEST inactivates E2 by E2S formation
In normal breast cells, a sulfotransferase (EST) is present that acts at nanomolar concentrations of E2 to form estradiol sulfate (E2 S) and consequently blocks the proliferative effect of E2 (E2 S is biologically inactive). However, in breast cancer cells, phenol sulfotransferase is active at micromolar concentrations of E2 (Figs. 7 and 8) and EST is not present [56–58]. As the progestin medrogestone stimulates EST in breast
Fig. 9. Hypothetical effects of medrogestone on estradiol sulfotransferase (EST) and proliferation in T-47D and MCF-7 breast cancer cells. As medrogestone can stimulate EST in the cancer cell, estradiol (E2 ) becomes inactive due to the formation of estradiol sulfate (E2 S) and consequently cell proliferation is inhibited.
Mechanism of Sulfotransferases (ST) activities in normal and breast cancer cells
I) Estrogen ST (EST) E2 E2S at nanomolar
II) Phenol ST (P-ST) E2 E2S at micromolar
(10 M) Normal Breast Cell
(10 M) Breast Cancer Cell
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Fig. 7. Mechanism of sulfotransferase (ST) activity in normal and breast cancer cells. In normal breast cells, it is suggested that hEST acts at physiological (nanomolar) concentrations of estradiol (E2 ) to form estradiol sulfate (E2 S), which is biologically inactive. This enzyme is absent from breast cancer cells, where phenol-ST activity is seen only at micromolar (non-physiological) concentrations.
III) Cell proliferation
cancer cells, and as it blocks the proliferation of T-47D cells, it has been suggested that the anti-proliferative effect of medrogestone is correlated with the stimulatory effect of human estrogen sulfotransferase in breast cancer cells (Fig. 9). More information on the proliferative effect on breast cancer cells of various progestins or other molecules at nanomolar concentrations is needed to verify this hypothesis.
6. Conclusions One of the possible ways of blocking the effect of E2 in breast cancer is by the use of anti-estrogens, which act by binding to the estrogen receptor. More than 15 years experience have shown that breast cancer patients treated with the anti-estrogen tamoxifen (Nolvadex) have a significantly reduced risk of recurrence and an increased overall survival. Recently, tests using a series of new anti-estrogens yielded very
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tion of estrogens in breast cancer can be summarised by the concept of the selective estrogen enzyme modulators (SEEM) (Fig. 10).
References
Fig. 10. The selective estrogen enzyme modulator (SEEM) concept in human hormone-dependent breast cancer cells. The SEEM can control enzymatic mechanisms involved in the formation and transformation of estrogens in breast cancer cells, where the sulfatase pathway is quantitatively higher than the aromatase pathway. SEEM-I inhibits estrone sulfatase, SEEM-II inhibits 17-hydroxysteroid dehydrogenase type 1, SEEM-III inhibits aromatase activities, and SEEM-IV stimulates estrone sulfotransferase activity. It is suggested that E1 S in tumours is present outside the cell and reaches the cell membrane where it is in contact with intracellular estrone sulfatase. ANDR.: androgens; E1 : estrone; E2 : estradiol; E1 S: estrone sulfate. From Chetrite and Pasqualini [59].
encouraging clinical results. Another way to block E2 is using anti-enzymes (anti-sulfatase, anti-aromatase or anti-17-HSD) against enzymes that are involved in E2 biosynthesis in breast cancer tissues. At present, anti-aromatase is extensively used in breast cancer treatment with positive benefits. However, estrone sulfatase is quantitatively the most important pathway in E2 bioformation in breast cancer tissue. Interesting data have been obtained concerning the inhibitory activity of various progestins (promegestone, nomegestrol acetate, medrogestone, dydrogesterone and its 20-dihydro derivative), as well as tibolone and its metabolites, on estrone sulfatase, as well as on 17-HSD, which is involved in another pathway of E2 formation in breast cancer cells. Recent data also show that some progestins (promegestone, nomegestrol acetate, dydrogesterone and medrogestone) and tibolone can stimulate sulfotransferase activity in hormone-dependent breast cancer cells. This is an important point in the physiopathology of this disease, as it is well known that estrogen sulfates are biologically inactive. The inhibitory and stimulatory effects on the control of enzymes involved in the formation and transforma-
[1] Loriaux DL, Ruder HJ, Lipsett MB. The measurement of estrone sulfate in plasma. Steroids 1971;18:463–73. [2] Santen RJ, Leszczynski D, Tilson-Mallet N, Feil PD, Wright C, Manni A, Santner SJ. Enzymatic control of estrogen production in human breast cancer: relative significance of aromatase vs. sulfatase pathway. In: Angeli A, Bradlow HL, Dogliotte L, editors. Endocrinology of the breast: basic and clinical aspects, vol. 464. New York, NY: Academic Press; 1986. p. 126–37. [3] Pasqualini JR, Chetrite G, Blacker M-C, Feinstein M-C, Delalonde L, Talbi M, Maloche C. Concentrations of estrone, estradiol, estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. J Clin Endocrinol Metab 1996;81:1460–4. [4] Vignon F, Terqui M, Westley B, Derocq D, Rochefort H. Effects of plasma estrogen sulfates in mammary cancer cells. Endocrinology 1980;106:1079–86. [5] Westley B, Rochefort H. A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 1980;20:353– 62. [6] Pasqualini JR, Gelly C, Lecerf F. Estrogen sulfates: biological and ultrastructural responses and metabolism in MCF-7 human breast cancer cell. Breast Cancer Res Treat 1986;8:233–40. [7] Pasqualini JR, Gelly C, Lecerf F. Biological effects and morphological responses to estriol-3-sulfate, estriol-17-sulfate and tamoxifen in a tamoxifen-resistant cell line (R-27) derived from MCF-7 human breast cancer cells. Eur J Cancer Clin Oncol 1986;22:1495–501. [8] Santner SJ, Ohlsson-Wilhelm B, Santen RJ. Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures. Eur J Cancer 1993;54:119–24. [9] Chetrite G, Cortes-Prieto J, Philippe J-C, Wright F, Pasqualini JR. Comparison of estrogen concentrations, estrone sulfatase and aromatase activities in normal, and in cancerous, human breast tissues. J Steroid Biochem Mol Biol 2000;72:23–7. [10] Naitoh K, Honjo H, Yamamoto T, Urabe M, Ogino Y, Yasumura T, Nambara T. Estrone sulfate and sulfatase activity in human breast cancer and endometrial cancer. J Steroid Biochem 1989;33:1049–54. [11] Imoto S, Mitani F, Enomoto K, Fujiwara K, Ikeda T, Kitajima M, Ishimura Y. Influence of estrogen metabolism on proliferation of human breast cancer. Breast Cancer Res Treat 1997;42:57–64. [12] Pasqualini JR, Cortes-Prieto J, Chetrite G, Talbi M, Ruiz A. Concentrations of estrone, estradiol and their sulfates, and evaluation of sulfatase and aromatase activities in patients with breast fibroadenoma. Int J Cancer 1997;70:343–69.
J.R. Pasqualini / Maturitas 46S1 (2003) S45–S54 [13] Pasqualini JR, Schatz B, Varin C, Nguyen B-L. Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer. J Steroid Biochem Mol Biol 1992;41: 323–9. [14] Pasqualini JR, Chetrite G, Nguyen B-L, Maloche C, Delalonde L, Talbi M, Feinstein M-C, Blacker C, Botella J, Paris J. Estrone sulfate-sulfatase and 17-hydroxysteroid dehydrogenase activities: a hypothesis for their role in the evolution of human breast cancer from hormone-dependence to hormone-independence. J Steroid Biochem Mol Biol 1995;53:407–12. [15] Pasqualini JR, Maloche C, Maroni M, Chetrite G. Effect of the progestagen promegestone (R-5020) on mRNA of the oestrone sulphatase in the MCF-7 human mammary cancer cells. Anticancer Res 1994;14:1589–94. [16] Pasqualini JR, Gelly C. Effect of tamoxifen and tamoxifen derivatives on the conversion of estrone-sulfate to estradiol in the MCF-7 and R-27 mammary cancer cell lines. Cancer Lett 1988;40:115–21. [17] Pasqualini JR, Giambiagi N, Gelly C, Chetrite G. Antiestrogen action in mammary cancer and in fetal cells. J Steroid Biochem Mol Biol 1990;37:343–8. [18] Pasqualini JR, Nguyen B-L. Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs. independent human breast cancer cell lines. Breast Cancer Res Treat 1991;18:93–8. [19] Santner SJ, Santen RJ. Inhibition of estrone sulfatase and 17-hydroxysteroid dehydrogenase by antiestrogens. J Steroid Biochem Mol Biol 1993;45:383–90. [20] Chu GH, Peters A, Selcer KW, Li A, Peters PK. Synthesis and sulfatase inhibitory activities of (E)- and (Z)-4-hydroxytamoxifen sulfamates. Bioorg Med Chem Lett 1999;18:141–4. [21] Pasqualini JR, Varin C, Nguyen B-L. Effect of the progestagen R5020 (promegestone) and of progesterone on the uptake and on the transformation of estrone sulfate in the MCF-7 and T-47D human mammary cancer cells: correlation with progesterone receptor levels. Cancer Lett 1992;66:55–60. [22] Chetrite G, Paris J, Botella J, Pasqualini JR. Effect of nomegestrol actetate on estrone sulfatase and 17-hydroxysteroid dehydrogenase activities in human breast cancer cells. J Steroid Biochem Mol Biol 1996;58:525–31. [23] Chetrite G, Ebert C, Wright F, Philippe J-C, Pasqualini JR. Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. J Steroid Biochem Mol Biol 1999;70:39–45. [24] Pasqualini JR, Caubel P, Friedman AJ, Philippe J-C, Chetrite GS. Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate. J Steroid Biochem Mol Biol 2003;84:193–198. [25] Pasqualini JR, Thole HH, Chetrite GS. Dydrogesterone (Duphaston® ) and its 20-dihydro-derivative as selective estrogen enzyme modulators in human breast cancer cell lines. Effects on sulfatase and on 17-hydroxysteroid dehydrogenase (17-HSD) activity. Anticancer Res. 2004, in press.
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[26] Chetrite GS, Kloosterboer HJ, Pasqualini JR. Effect of tibolone (Org OD14) and its metabolites on estrone sulphatase activity in MCF-7 and T-47D mammary cancer cells. Anticancer Res 1997;17:135–40. [27] Selcer KW, Li P-K. Estrogenicity, antiestrogenecity and estrone sulfatase inhibition of estrone-3-amine and estrone3-thiol. J Steroid Biochem Mol Biol 1995;52:281–6. [28] Howarth NM, Purohit A, Reed MJ, Potter BVL. Estrone sulfamates: potent inhibitors of estrone sulfatase with therapeutic potential. J Med Chem 1994;37:219–21. [29] Purohit A, Williams GJ, Roberts CJ, Potter BVL, Reed MJ. In vivo inhibition of oestrone sulphatase and dehydroepiandrosterone sulphatase by oestrone-3-O-sulphamate. Int J Cancer 1995;63:106–11. [30] Elger W, Schwarz S, Hedden A, Reddersen G, Schneider B. Sulfamates of various estrogens are prodrugs with increased systemic and reduced hepatic estrogenicity at oral application. J Steroid Biochem Mol Biol 1995;55:395– 403. [31] Valigora SD, Li P-K, Dunphy G, Turner M, Ely DL. Steroid sulfatase inhibitor alters blood pressure and steroid profiles in hypertensive rats. J Steroid Biochem Mol Biol 2000;73:113– 22. [32] Anderson CJ, Lucas LJH, Widlanski TS. Molecular recognition in biological systems: phosphate esters vs. sulfate esters and the mechanism of action of steroid sulfatase. J Am Chem Soc 1995;117:3889–90. [33] Boivin RP, Luu-The V, Lachance R, Labrie F, Poirier D. Structure-activity relationships of 17alpha-derivates of estradiol as inhibitors of steroid-sulfatase. J Med Chem 2000;43:4465–78. [34] Poirier D, Boivin RP. 17␣-Alkyl- or 17␣-substituted benzyl17-estradiols: a new family of estrone-sulfatase inhibitors. Bioorg Med Chem Lett 1998;8:1891–6. [35] Ciobanu LC, Boivin RP, Luu-The V, Labrie F, Poirier D. Potent inhibition of steroid sulfatase activity by 3-O-sulfamate 17␣-benzyl (or 4-tert-butylbenzyl)estra-1,3,5(10)-trienes: combination of two substituents at positions C3 and C17␣ of estradiol. J Med Chem 1999;42:2280–6. [36] Purohit A, Vernon KA, Hummelinck AEW, Woo LW, Hejaz HA, Potter BV, Reed MJ. The development of a-ring modified analogues of oestrone-3-O-sulphamate as potent steroid sulphatase inhibitors with reduce oestrogenecity. J Steroid Biochem Mol Biol 1998;64:269–76. [37] Purohit A, Woo LWL, Barrow D, Hejaz HA, Nicholson RI, Potter BV, Reed MJ. Non-steroidal and steroidal sulfamates: new drugs for cancer therapy. Mol Cell Endocr 2001;171:129– 35. [38] Malini B, Purohit A, Ganeshapillai D, Woo LWL, Potter BVL, Reed MJ. Inhibition of steroid sulphatase activity by tricyclic coumarin sulphamates. J Steroid Biochem Mol Biol 2000;75:253–8. [39] Woo LWL, Lightowler M, Purohit A, Reed MJ, Potter BVL. Heteroatom-substituted analogues of the active-site directed inhibitor estra-1,3,5(10)-trien-17-one-3-sulphamate inhibit estrone sulphatase by a different mechanism. J Steroid Biochem Mol Biol 1996;57:79–88.
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[40] Woo LWL, Purohit A, Malini B, Reed MJ, Potter BVL. Potent active site-directed inhibition of steroid sulphatase by tricyclic coumarin-based sulphamates. Chem Biol 2000;7:773– 91. [41] Pasqualini JR, Chetrite G. Paradoxical effect of estradiol: it can block its own bioformation in human breast cancer cells. J Steroid Biochem Mol Biol 2001;78:21–4. [42] Pollow K, Boquoi E, Baumann J, Schmidt-Gollwitzer M, Pollow B. Comparison of the in vitro conversion of estradiol-17 to estrone of normal and neoplastic human breast tissue. Mol Cell Endocr 1977;6:333–48. [43] Miettinen M, Mustonen M, Poutanen M, Isomaa V, Wickman M, Soderqvist G, Vihko R, Vihko P. 17-hydroxysteroid dehydrogenases in normal human mammary epithelial cells and breast tissue. Breast Cancer Res Treat 1999;57:175–82. [44] Poutanen M, Moncharmont B, Vihko R. 17-Hydroxysteroid dehydrogenase gene expression in human breast cancer cells: regulation of expression by a progestin. Cancer Res 1992;52:290–4. [45] Bonney RC, Reed MJ, Davidson K, Beranek PA, James VHT. The relationship between 17-hydroxysteroid dehydrogenase activity and oestrogen concentrations in human breast tumours and in normal breast tissue. Clin Endocr 1983;19:727–39. [46] Couture P, Theriault C, Simard J, Labrie F. Androgen receptor-mediated stimulation of 17-hydroxysteroid dehydrogenase activity by dihydrotestosterone and medroxyprogesterone acetate in ZR-75-1 human breast cancer cells. Endocrinology 1993;132:179–85. [47] Nguyen B-L, Chetrite G, Pasqualini JR. Transformation of estrone and estradiol in hormone-dependent and hormoneindependent human breast cancer cells. Breast Cancer Res Treat 1995;34:139–46. [48] Peltoketo H, Isomaa VV, Poutanen MH, Vihko R. Expression and regulation of 17-hydroxysteroid dehydrogenase type 1. J Endocrinol 1996;150:S21–30. [49] Prudhomme JF, Malet C, Gompel A, Lalardie JP, Ochoa C, Boue A, Mauvais-Jarvis P, Kuttenn F. 17-Hydroxysteroid dehydrogenase activity in human breast epithelial cells and fibroblast cultures. Endocrinology 1984;114:1483–9.
[50] Gompel A, Malet C, Spritzer P, Lalardrie J-P, Kuttenn F, Mauvais-Jarvis P. Progestin effect on cell proliferation and 17-hydroxysteroid dehydrogenase activity in normal human breast cells in culture. J Clin Endocrinol Metab 1986;63:1174–80. [51] Coldham NG, James VHT. A possible mechanism for increased breast cell proliferation by progestins through increased reductive 17-hydroxysteroid dehydrogenase activity. Int J Cancer 1990;45:174–8. [52] Adams EF, Coldham NG, James VHT. Steroidal regulation of oestradiol-17-dehydrogenase activity of the human breast cancer cell line MCF-7. J Endocrinol 1988;118:149–54. [53] McNeill JM, Reed MJ, Beranek PA, Newton CJ, Ghilchik MW, James VHT. The effect of epidermal growth factor, transforming growth factor and breast tumour homogenates on the activity of oestradiol 17-hydroxysteroid dehydrogenase in cultured adipose tissue. Cancer Lett 1986;31:213–9. [54] Malet C, Vacca A, Kuttenn F, Mauvais-Jarvis P. 17-Estradiol dehydrogenase (E2DH) activity in T47D cells. J Steroid Biochem Mol Biol 1991;39:769–75. [55] Chetrite GS, Ebert C, Wright F, Philippe J-C, Pasqualini JR. Effect of medrogestone on 17-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. J Steroid Biochem Mol Biol 1999;68:51–6. [56] Qian Y, Deng C, Song W-C. Expression of estrogen sulfotransferase in MCF-7 cells by cDNA transfection suppresses the estrogen response: potential role of the enzyme in regulating estrogen-dependent growth of breast epithelial cells. J Pharmacol Exp Ther 1988;2:555–60. [57] Falany JL, Falany CN. Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines. Cancer Res 1996;56:1551–5. [58] Falany JL, Falany CN. Regulation of estrogen activity by sulfation in human MCF-7 human breast cancer cells. Oncol Res 1997;9:589–96. [59] Chetrite GS, Pasqualini JR. The selective estrogen enzyme modulator (SEEM) in breast cancer. J Steroid Biochem Mol Biol 2001;76:95–104.
Differential effects of progestins on breast tissue enzymes J.R. Pasqualini∗ Hormones and Cancer Research Unit, Institut de Puériculture, 26 Blvd. Brune, Paris 75014, France
Abstract There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E2 ) from circulating precursors. Two principal pathways are implicated in the final steps of E2 formation in breast cancer tissue: the ‘aromatase pathway’ that transforms androgens into estrogens and the ‘sulfatase pathway’ that converts estrone sulfate (E1 S) into estrone (E1 ) via estrone sulfatase. The final step is the conversion of weak E1 to potent biologically active E2 via reductive 17-hydroxysteroid dehydrogenase type 1 activity. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are present in breast cancer tissues. One of the possible means of blocking E2 effects in breast cancer is to use anti-estrogens, which act by binding to the estrogen receptor (ER). Another option is to block E2 using anti-enzymes (anti-sulfatase, anti-aromatase, or anti-17-hydroxysteroid dehydrogenase (17-HSD). Various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, 17-deacetyl norgestimate, dydrogesterone and its 20-dihydro derivative), as well as tibolone and its metabolites, have been shown to inhibit estrone sulfatase and 17-hydroxysteroid dehydrogenase. Some progestins and tibolone can also stimulate sulfotransferase activity. These various progestins may therefore provide a new option for the treatment of breast cancer. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: 17-Hydroxysteroid dehydrogenase; Progestin; Breast cancer
1. Introduction The “intracrine concept”, where a hormone has a biological response in the same organ in which it is produced, is entirely applicable to breast carcinoma tissue for two main reasons: (1) breast cancer tissue accumulates extensive quantities of estrogens (unconjugates and sulfoconjugates), particularly in postmenopausal patients; (2) the enzyme systems for the bioformation and metabolic transformation of estrogens are present in high levels in this tissue. It is clear that the biological responses to steroid hor∗
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mones in their target tissue are subject to modulation by a variety of enzymes. These enzymes may control the access of steroids to their receptors by converting them into forms with a higher affinity for the receptor or by inactivating the corresponding steroid. One of the possible ways of blocking the estradiol (E2 ) effect in breast cancer is via anti-estrogens. Another way to block E2 is by using anti-enzymes (anti-sulfatase, anti-aromatase, or anti-17-hydroxysteroid dehydrogenase (17-HSD) (type 1 against the enzymes that are involved in E2 biosynthesis in breast cancer tissues)). At present, anti-aromatases are extensively used in breast cancer treatment with positive benefits. However, estrone sulfatase is quantitatively the most important pathway in E2 bioformation in
0378-5122/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2003.09.018
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breast cancer tissue. In this review, we summarise the effect of various progestins on estrone sulfatase, 17-hydroxysteroid dehydrogenase and estrone sulfotransferase in human breast cancer tissue.
2. Estrone sulfatase and its control in breast cancer 2.1. The importance of estrone sulfatase in breast cancer E2 is one of the most important factors in the growth and evolution of hormone-dependent breast tumors and it is now well accepted that breast tumors have the capacity to locally synthesize active E2 . Two metabolic pathways are implicated in this intra-tumoral biosynthesis: the “aromatase pathway” that converts adrenal androgen precursors (e.g. androstenedione) to estrone (E1 ) by aromatisation, followed by reduction of estrone to E2 via 17-hydroxysteroid dehydrogenase (17-HSD) type 1; and the “sulfatase pathway” that transforms the precursor estrone sulfate (E1 S) to E1 via sulfatase in adipose tissues, followed by 17-HSD type 1 reductive activity to E2 . Estrogen sulfates and sulfatases are important in breast cancer due to the following reasons: 1. Estrone sulfate is the most abundant circulating estrogen in the plasma of postmenopausal women. The levels of this conjugate are 2–10 times higher than those of unconjugated E1 and E2 [1–3]. 2. As estrogen sulfates do not bind to estrogen receptors (ER), estrogen-3-sulfates must be hydrolyzed by sulfatases to elicit a biological response. E1 , after conversion to E2 , increases the amount of progesterone receptors (PR) and pS2 protein, as well as estrogen-inducible protein cathepsin D [4–8] in hormone-dependent breast cancer cells (MCF-7), whereas estrogen-17-sulfates, which are not hydrolyzed by sulfatases, do not cause any biological responses [6,7]. 3. The incidence of breast cancer is higher after the menopause when the ovaries have ceased to be functional and the levels of E2 and estrone sulfate are 7–11 times higher in breast tumour tissue than in plasma [3].
4. The concentration of estrone sulfate in the tumor is higher in postmenopausal than in premenopausal breast cancer patients [3]. 5. The tissue concentration of estrone sulfate is higher in tumoral than in normal breast tissue [9]. 6. Sulfatase activity is very intense in malignant and benign breast tumors compared with normal breast tissue [9–12]. 7. Quantitative measurements indicate that levels of estrone sulfatase are 40–500 times higher than those of aromatase in breast tumour tissue [3]. 2.2. Control of estrone sulfatase in breast cancer Estrone sulfatase activity is high in human hormone-dependent breast cancer cells (MCF-7, T-47D). In contrast, hormone-independent breast cancer cells (MDA-MB-231, MDA-MB-468) show very low sulfatase activity in intact cells, but the activity is restored when the cells are homogenized [13,14]. The sulfatase mRNA is present in both the hormone-dependent and hormone-independent breast cancer cells, and the expression of this mRNA correlates with sulfatase activity [15]. The data clearly indicate that sulfatase is present in hormone-independent cells, but that it does not function in complete cells. Why, in spite of the existence of the enzyme, is very little estrone sulfate hydrolyzed within these intact cells? The answer to this question is not currently clear, but suggestions include the presence of repressive factor(s) or sequestration into an inactive form for this kind of cell. More information is needed to elucidate this mechanism. As estrone sulfatase is the other important means of producing biologically active estrogens, blockade of the sulfatase pathway constitutes a promising alternative for reducing the level of E2 . In recent years, the potential inhibitory effects of a wide range of compounds, including anti-estrogens, progestins, tibolone and its metabolites, as well as steroidal and non-steroidal compounds, have been explored. 2.2.1. Inhibition by anti-estrogens The anti-estrogen tamoxifen and its more potent metabolite, 4-hydroxytamoxifen, as well as ICI 164,384, have been reported to inhibit sulfatase activity, probably by a non-competitive mechanism [16–19]. Chu et al. [20] found that, in the rat liver,
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Fig. 1. Comparative effects of various progestins on inhibition of estrone sulfate (E1 S) conversion to estradiol (E2 ) in hormone-dependent T-47D human breast cancer cells. The values represent mean ± S.E.M. of duplicate determinations of three–seven independent experiments. R-5020: promegestone; Nom. Ac.: nomegestrol acetate; Medrog.: medrogestone; Noreth.: norethisterone. ∗ P ≤ 0.05 vs. control value; ∗∗ P ≤ 0.01 vs. control value. From Chetrite and Pasqualini [59].
(E)- and (Z)-4-hydroxytamoxifen sulfamates are also sulfatase inhibitors, with Ki values of 35.9 and >500 M, respectively. 2.2.2. Inhibition by progestins Incubation of physiological concentrations (5 × 10−9 M) of estrone sulfate with MCF-7 or T-47D human breast cancer cell lines showed that various progestins (nomegestrol acetate, promegestone, progesterone, medrogestone (Prothil® ), 17-deacetyl norgestimate can provoke significant inhibition of estrone sulfatase activity [13,21–24]. Fig. 1 shows the relative percentage of sulfatase inhibition by theseprogestins. Recently, it was demonstrated that dydrogesterone (Duphaston® ) and its 20-dihydro derivatives are potent inhibitors of estrone sulfatase in MCF-7 and T-47D breast cancer cells [25]. This is indicated in Fig. 2. 2.2.3. Inhibition by tibolone and its metabolites Tibolone (Org OD14; active substance of Livial® (2.5 mg)) is a 19-nortestosterone derivative with tissue-specific estrogenic, progestogenic and androgenic properties. It is used as monotherapy for the treatment of climacteric symptoms and the prevention of osteoporosis, without stimulating the endometrium. Tibolone, and its more active metabolites Org 4094, Org 30,126 (3␣- and 3-hydroxy derivatives) and
Org OM38 (the 4-en isomer), are potent sulfatase inhibitors at low concentrations (5 × 10−7 M) in MCF-7 and T-47D hormone-dependent breast cancer cells [26].
Fig. 2. Effects of dydrogesterone (DYD) and 20-dihydro derivate (DHD) on the conversion of estrone sulfate (E1 S) to estradiol (E2 ) in the hormone-dependent MCF-7 human breast cancer cell line. Cells were incubated for 24 h at 37 ◦ C with a physiological concentration (5 × 10−9 mol/l) of [3 H]-E1 S alone (control: non-treated cells) or in the presence of DYD or DHD at the range of concentrations from 5 × 10−9 mol/l to 5 × 10−5 mol/l. Qualitative and quantitative analyses of E2 in the cell compartment were performed by thin-layer chromatography and liquid scintillation counting. Results are expressed in fmol of E2 formed/mg DNA from E1 S. The data are the mean ± S.E.M. of duplicate determinations of three independent experiments. ∗ P = 0.05 vs. control values (non-treated cells).
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2.2.4. Inhibition by steroidal compounds Estrone-3-O-sulfamate (EMATE) is a potent synthesized sulfatase inhibitor [27]; at a concentration of 10−7 M, estrone sulfatase activity in MCF-7 cells is 99% inhibited [28,29]. Unfortunately, the potent estrogenic activity of this compound precludes its use in clinical applications [30,31]. Estrone phosphate and DHEA-phosphate are also potent inhibitors of estrogen sulfatase activity [32]. In other studies, Boivin et al. [33] and Poirier and Boivin [34] attempted to develop sulfatase inhibitors without residual estrogenic activity by synthesizing a series of E2 derivatives bearing an alkyl, a phenyl, a benzyl substituted (or not), or an alkyan amide side chain at position 17␣. These studies showed that sulfatase inhibitors act by a reversible mechanism and that the hydrophobic group at the 17␣ position increases the inhibitory activity, whilst stearic factors contribute to the opposite effect. The most potent inhibitor is a 17␣-benzyl substituted E2 derivative with an IC50 value of 22 nM. When these 17␣-substituents were added to the 3-O-sulfamate estradiol structure, the combined inhibitory effect was more potent, with an IC50 value of 0.15 nM [35]. 2.2.5. Inhibition by non-steroidal compounds An interesting new family of compounds has been synthesized with a tricyclic coumarin sulfamate structure [36–40]. These non-steroidal sulfatase inhibitors are active in vitro and in vivo, are non-estrogenic and possess, in vitro, an IC50 value of approximately 1 nM. However, the most potent inhibitor in vivo does not correspond to the best compound in vitro. 2.2.6. Inhibition of estrone sulfatase activity by estradiol (E2 ) Recent studies have demonstrated a paradoxical effect of E2 in MCF-7 and T-47D breast cancer cells in that it can block its own bioformation by inhibiting, in a dose-dependent manner, the conversion of estrone sulfate to E2 at concentrations of 5 × 10−10 M to 5 × 10−5 M [41] (Fig. 3). E2 is a potent inhibitor of estrone sulfatase activity, with IC50 values of 1.84 × 10−9 M and 8.77 × 10−10 M in T-47D and MCF-7 cells, respectively.
Fig. 3. Effect of estradiol (E2 ) on the conversion of estrone sulfate (E1 S) to E2 in MCF-7 human breast cancer cells. The percentage of inhibition was obtained by calculating the ratio [(control − test)/control] × 100. The values represent mean ± S.E.M. of duplicate determinations of five independent experiments. ∗ P ≤ 0.05 vs. control value; ∗∗ P ≤ 0.005 vs. control value. From Pasqualini and Chetrite [41].
3. 17-Hydroxysteroid dehydrogenase in the breast 17-Hydroxysteroid dehydrogenase (17-HSD), a widely distributed enzyme in mammalian tissues, is implicated in the inter-conversion of the inactive 17-keto to the active 17-hydroxy form of sex steroid hormones (estrogens and androgens). However, some types of 17-HSD may metabolize other substrates such as bile acids, alcohols, fatty acids and retinols. 17-HSD belongs to a superfamily of enzymes in which 11 different isoforms have been recognised to date. In normal breast tissue, oxidative 17-HSD activity (E2 to E1 ) was found to be the preferential direction and this activity was more intense during the secretory phase of the menstrual cycle [42]; 17-HSD type 1 and 2 mRNA are both expressed in glandular epithelium. In HME human mammary epithelial cell lines, mRNA for 17-HSD types 1, 2, and 4 was detected, but only oxidative 17-HSD activity was present; it
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was suggested that this activity is due to 17-HSD type 2 [43]. In breast tumours, in vivo and in vitro studies show that the preferential conversion is the reduction of E1 to E2 . 17-HSD type 1 is found in the cytoplasm of malignant epithelial cells in breast tumours [44]. However, the direction of enzymatic activity (oxidative or reductive) in breast cancer is also greatly dependent on the local, metabolic or experimental conditions, including: the nature and concentration of the cofactors (e.g. NADPH or NADP) and substrate, pH, and the subcellular localisation of enzymes. In vitro studies using human tumour homogenates indicated that the predominant 17-HSD activity was oxidative rather than reductive [45]. However, in vivo studies following isotopic infusion of estrogens in postmenopausal breast cancer patients have shown that the reductive direction is greater than the oxidative [44]. In hormone-dependent breast cancer cell lines (MCF-7, T-47D, R-27, ZR-75-1), 17-HSD type 1 was found to be the predominant reductive isoform, but types 2 and 4 isoforms with oxidative activities (formation of E1 ) were also detected [44,46–48]. 3.1. Control of 17β-HSD in the breast In epithelial cells from normal breast, the progestin promegestone (R-5020) was shown to increase 17-HSD activity in the oxidative (E2 to E1 ) direc-
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tion; this stimulatory effect of progestins depends on preliminary sensitization by estrogens [49,50]. Progestins can induce 17-HSD type 1 activity, with an increase in both the 1.3 kb mRNA species and the enzyme protein, in hormone-dependent T-47D breast cancer cells [44,48]. Coldham and James [51] found that the progestin medroxyprogesterone acetate (MPA) stimulated the reductive (E1 to E2 ) activity of MCF-7 cells when phenol red was excluded from the tissue culture media. The authors suggested that this could be the way in which progestins increase cell proliferation in vivo. On the other hand, Couture et al. [46] observed that when hormone-dependent ZR-75-1 breast cancer cells were incubated with MPA, the oxidative (E2 to E1 ) direction was predominant; this effect seems to implicate the androgen receptor. Other progestins, such as progesterone, levonorgestrel and norethisterone, increased both the oxidative and reductive 17-HSD activity in MCF-7 cells [52], whereas promegestone (R-5020) had no significant effect on the reductive activity of 17-HSD [53] but increased the oxidative (E2 to E1 ) activity in T-47D cells [54]. Nomegestrol acetate had an inhibitory effect on the 17-HSD enzyme in T-47D cells (35 and 81% inhibition at 5 × 10−7 M and 5 × 10−6 M, respectively), but no significant effect was found in MCF-7 cells, except at 5 × 10−5 M [22]. Medrogestone (Prothil® ), a synthetic pregnane derivative of progesterone, significantly decreased the reductive 17-HSD type 1 activity in MCF-7 and T-47D breast cancer cells. The
Fig. 4. Comparative effects of various progestins on inhibition of the conversion of estrone (E1 ) to estradiol (E2 ) in hormone-dependent T-47D human breast cancer cells. The values represent mean ± S.E.M. of duplicate determinations of three–six independent experiments. R-5020: promegestone; Danaz.: danazol; Medrog.: medrogestone; Nom. Ac.: nomegestrol acetate. ∗ P ≤ 0.05 vs. control value. From Chetrite and Pasqualini [59].
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inhibitory effect was concentration-dependent and more intense, even at low concentrations, in the T-47D cell line than in the MCF-7 cells; the IC50 values, which correspond to 50% inhibition of the conversion of E1 to E2 , were 0.45 and 17.36 M, respectively [55]. Comparative effects of various progestins: promegestone, danazol, medrogestone, nomegestrol acetate, on the inhibitory effect of 17-HSD in T-47D cells are indicated in Fig. 4. Recently, it was shown that dydrogesterone (Duphaston® ) and its 20-dihydro derivatives can also inhibit the conversion of E1 to E2 in the T-47D breast cancer cells (Fig. 5). Fig. 5. Effects of dydrogesterone (DYD) and its 20-dihydro derivate (DHD) on the conversion of estrone (E1 ) to estradiol (E2 ) in the hormone-dependent T-47D human breast cancer cell line. Cells were incubated for 24 h at 37 ◦ C with a physiological concentration (5 × 10−9 mol/l) of [3 H]-E1 alone (control: non-treated cells) or in the presence of DYD or DHD at the range of concentrations from 5 × 10−9 mol/l to 5 × 10−5 mol/l. Qualitative and quantitative analyses of E2 in the cell compartment were performed by thin-layer chromatography and liquid scintillation counting. Results are expressed in fmol of E2 formed/mg DNA from E1 . The data are the mean ± S.E.M. of duplicate determinations of three independent experiments. ∗ P = 0.05 vs. control values (non-treated cells).
4. Sulfotransferase and its control in breast cancer As sulfoconjugates are not biologically active, control of their formation in breast cells represents an important mechanism by which to modulate the biological action of E2 in this tissue. Comparative studies on the formation of estrogen sulfates after incubation of E1 with hormone-dependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231) breast cancer
Fig. 6. Effects of medrogestone (Prothil® ) on the conversion of estrone (E1 ) to estrogen sulfates in hormone-dependent MCF-7 and T-47D human breast cancer cells. Results (pmol of ES formed/mg DNA) are expressed in percent of control values considered as 100%. The values represent mean ± S.E.M. of duplicate determinations of three independent experiments. ∗ P ≤ 0.05 vs. control value (non-treated cells). ∗∗ P ≤ 0.01 vs. control value (non-treated cells). From Chetrite et al. [23].
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cells show significantly higher sulfotransferase levels in the former [55]. 4.1. Effect of medrogestone and other progestins Medrogestone is a synthetic pregnane derivative used in the treatment of pathological deficiency of natural progesterone. This compound has secretory activity in the estrogen-primed uterus, is thermogenic and acts as an anti-estrogen and antigonadotropin. In MCF-7 and T-47D breast cancer cells, medrogestone had a bi-phasic effect on sulfotransferase activity: at a low concentration (5 × 10−8 mol/l) it stimulated the formation of estrogen sulfates in both cells lines, whereas at a high concentration (5 × 10−5 mol/l) sulfotransferase activity was not modified in MCF-7 cells or inhibited in T-47D cells [23] (Fig. 6).
Fig. 8. Effects of estradiol sulfotransferase (EST) activity on the proliferation of breast cancer cells. In normal breast cells, as a consequence of EST activity, proliferation is inhibited because estradiol sulfate (E2 S) is biologically inactive. In contrast, in breast cancer cells, hEST activity is very low or non-existent as E2 S is not formed and E2 can stimulate proliferation.
Hypothetical effect of Medrogestone on Sulfotransferase (hEST) and Proliferation in MCF-7 cells I) MEDROGESTONE stimulates hEST
5. Possible correlation of sulfotransferase activity and proliferation of breast cancer cells
II) hEST inactivates E2 by E2S formation
In normal breast cells, a sulfotransferase (EST) is present that acts at nanomolar concentrations of E2 to form estradiol sulfate (E2 S) and consequently blocks the proliferative effect of E2 (E2 S is biologically inactive). However, in breast cancer cells, phenol sulfotransferase is active at micromolar concentrations of E2 (Figs. 7 and 8) and EST is not present [56–58]. As the progestin medrogestone stimulates EST in breast
Fig. 9. Hypothetical effects of medrogestone on estradiol sulfotransferase (EST) and proliferation in T-47D and MCF-7 breast cancer cells. As medrogestone can stimulate EST in the cancer cell, estradiol (E2 ) becomes inactive due to the formation of estradiol sulfate (E2 S) and consequently cell proliferation is inhibited.
Mechanism of Sulfotransferases (ST) activities in normal and breast cancer cells
I) Estrogen ST (EST) E2 E2S at nanomolar
II) Phenol ST (P-ST) E2 E2S at micromolar
(10 M) Normal Breast Cell
(10 M) Breast Cancer Cell
-9
-6
Fig. 7. Mechanism of sulfotransferase (ST) activity in normal and breast cancer cells. In normal breast cells, it is suggested that hEST acts at physiological (nanomolar) concentrations of estradiol (E2 ) to form estradiol sulfate (E2 S), which is biologically inactive. This enzyme is absent from breast cancer cells, where phenol-ST activity is seen only at micromolar (non-physiological) concentrations.
III) Cell proliferation
cancer cells, and as it blocks the proliferation of T-47D cells, it has been suggested that the anti-proliferative effect of medrogestone is correlated with the stimulatory effect of human estrogen sulfotransferase in breast cancer cells (Fig. 9). More information on the proliferative effect on breast cancer cells of various progestins or other molecules at nanomolar concentrations is needed to verify this hypothesis.
6. Conclusions One of the possible ways of blocking the effect of E2 in breast cancer is by the use of anti-estrogens, which act by binding to the estrogen receptor. More than 15 years experience have shown that breast cancer patients treated with the anti-estrogen tamoxifen (Nolvadex) have a significantly reduced risk of recurrence and an increased overall survival. Recently, tests using a series of new anti-estrogens yielded very
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tion of estrogens in breast cancer can be summarised by the concept of the selective estrogen enzyme modulators (SEEM) (Fig. 10).
References
Fig. 10. The selective estrogen enzyme modulator (SEEM) concept in human hormone-dependent breast cancer cells. The SEEM can control enzymatic mechanisms involved in the formation and transformation of estrogens in breast cancer cells, where the sulfatase pathway is quantitatively higher than the aromatase pathway. SEEM-I inhibits estrone sulfatase, SEEM-II inhibits 17-hydroxysteroid dehydrogenase type 1, SEEM-III inhibits aromatase activities, and SEEM-IV stimulates estrone sulfotransferase activity. It is suggested that E1 S in tumours is present outside the cell and reaches the cell membrane where it is in contact with intracellular estrone sulfatase. ANDR.: androgens; E1 : estrone; E2 : estradiol; E1 S: estrone sulfate. From Chetrite and Pasqualini [59].
encouraging clinical results. Another way to block E2 is using anti-enzymes (anti-sulfatase, anti-aromatase or anti-17-HSD) against enzymes that are involved in E2 biosynthesis in breast cancer tissues. At present, anti-aromatase is extensively used in breast cancer treatment with positive benefits. However, estrone sulfatase is quantitatively the most important pathway in E2 bioformation in breast cancer tissue. Interesting data have been obtained concerning the inhibitory activity of various progestins (promegestone, nomegestrol acetate, medrogestone, dydrogesterone and its 20-dihydro derivative), as well as tibolone and its metabolites, on estrone sulfatase, as well as on 17-HSD, which is involved in another pathway of E2 formation in breast cancer cells. Recent data also show that some progestins (promegestone, nomegestrol acetate, dydrogesterone and medrogestone) and tibolone can stimulate sulfotransferase activity in hormone-dependent breast cancer cells. This is an important point in the physiopathology of this disease, as it is well known that estrogen sulfates are biologically inactive. The inhibitory and stimulatory effects on the control of enzymes involved in the formation and transforma-
[1] Loriaux DL, Ruder HJ, Lipsett MB. The measurement of estrone sulfate in plasma. Steroids 1971;18:463–73. [2] Santen RJ, Leszczynski D, Tilson-Mallet N, Feil PD, Wright C, Manni A, Santner SJ. Enzymatic control of estrogen production in human breast cancer: relative significance of aromatase vs. sulfatase pathway. In: Angeli A, Bradlow HL, Dogliotte L, editors. Endocrinology of the breast: basic and clinical aspects, vol. 464. New York, NY: Academic Press; 1986. p. 126–37. [3] Pasqualini JR, Chetrite G, Blacker M-C, Feinstein M-C, Delalonde L, Talbi M, Maloche C. Concentrations of estrone, estradiol, estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. J Clin Endocrinol Metab 1996;81:1460–4. [4] Vignon F, Terqui M, Westley B, Derocq D, Rochefort H. Effects of plasma estrogen sulfates in mammary cancer cells. Endocrinology 1980;106:1079–86. [5] Westley B, Rochefort H. A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 1980;20:353– 62. [6] Pasqualini JR, Gelly C, Lecerf F. Estrogen sulfates: biological and ultrastructural responses and metabolism in MCF-7 human breast cancer cell. Breast Cancer Res Treat 1986;8:233–40. [7] Pasqualini JR, Gelly C, Lecerf F. Biological effects and morphological responses to estriol-3-sulfate, estriol-17-sulfate and tamoxifen in a tamoxifen-resistant cell line (R-27) derived from MCF-7 human breast cancer cells. Eur J Cancer Clin Oncol 1986;22:1495–501. [8] Santner SJ, Ohlsson-Wilhelm B, Santen RJ. Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures. Eur J Cancer 1993;54:119–24. [9] Chetrite G, Cortes-Prieto J, Philippe J-C, Wright F, Pasqualini JR. Comparison of estrogen concentrations, estrone sulfatase and aromatase activities in normal, and in cancerous, human breast tissues. J Steroid Biochem Mol Biol 2000;72:23–7. [10] Naitoh K, Honjo H, Yamamoto T, Urabe M, Ogino Y, Yasumura T, Nambara T. Estrone sulfate and sulfatase activity in human breast cancer and endometrial cancer. J Steroid Biochem 1989;33:1049–54. [11] Imoto S, Mitani F, Enomoto K, Fujiwara K, Ikeda T, Kitajima M, Ishimura Y. Influence of estrogen metabolism on proliferation of human breast cancer. Breast Cancer Res Treat 1997;42:57–64. [12] Pasqualini JR, Cortes-Prieto J, Chetrite G, Talbi M, Ruiz A. Concentrations of estrone, estradiol and their sulfates, and evaluation of sulfatase and aromatase activities in patients with breast fibroadenoma. Int J Cancer 1997;70:343–69.
J.R. Pasqualini / Maturitas 46S1 (2003) S45–S54 [13] Pasqualini JR, Schatz B, Varin C, Nguyen B-L. Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer. J Steroid Biochem Mol Biol 1992;41: 323–9. [14] Pasqualini JR, Chetrite G, Nguyen B-L, Maloche C, Delalonde L, Talbi M, Feinstein M-C, Blacker C, Botella J, Paris J. Estrone sulfate-sulfatase and 17-hydroxysteroid dehydrogenase activities: a hypothesis for their role in the evolution of human breast cancer from hormone-dependence to hormone-independence. J Steroid Biochem Mol Biol 1995;53:407–12. [15] Pasqualini JR, Maloche C, Maroni M, Chetrite G. Effect of the progestagen promegestone (R-5020) on mRNA of the oestrone sulphatase in the MCF-7 human mammary cancer cells. Anticancer Res 1994;14:1589–94. [16] Pasqualini JR, Gelly C. Effect of tamoxifen and tamoxifen derivatives on the conversion of estrone-sulfate to estradiol in the MCF-7 and R-27 mammary cancer cell lines. Cancer Lett 1988;40:115–21. [17] Pasqualini JR, Giambiagi N, Gelly C, Chetrite G. Antiestrogen action in mammary cancer and in fetal cells. J Steroid Biochem Mol Biol 1990;37:343–8. [18] Pasqualini JR, Nguyen B-L. Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs. independent human breast cancer cell lines. Breast Cancer Res Treat 1991;18:93–8. [19] Santner SJ, Santen RJ. Inhibition of estrone sulfatase and 17-hydroxysteroid dehydrogenase by antiestrogens. J Steroid Biochem Mol Biol 1993;45:383–90. [20] Chu GH, Peters A, Selcer KW, Li A, Peters PK. Synthesis and sulfatase inhibitory activities of (E)- and (Z)-4-hydroxytamoxifen sulfamates. Bioorg Med Chem Lett 1999;18:141–4. [21] Pasqualini JR, Varin C, Nguyen B-L. Effect of the progestagen R5020 (promegestone) and of progesterone on the uptake and on the transformation of estrone sulfate in the MCF-7 and T-47D human mammary cancer cells: correlation with progesterone receptor levels. Cancer Lett 1992;66:55–60. [22] Chetrite G, Paris J, Botella J, Pasqualini JR. Effect of nomegestrol actetate on estrone sulfatase and 17-hydroxysteroid dehydrogenase activities in human breast cancer cells. J Steroid Biochem Mol Biol 1996;58:525–31. [23] Chetrite G, Ebert C, Wright F, Philippe J-C, Pasqualini JR. Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. J Steroid Biochem Mol Biol 1999;70:39–45. [24] Pasqualini JR, Caubel P, Friedman AJ, Philippe J-C, Chetrite GS. Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate. J Steroid Biochem Mol Biol 2003;84:193–198. [25] Pasqualini JR, Thole HH, Chetrite GS. Dydrogesterone (Duphaston® ) and its 20-dihydro-derivative as selective estrogen enzyme modulators in human breast cancer cell lines. Effects on sulfatase and on 17-hydroxysteroid dehydrogenase (17-HSD) activity. Anticancer Res. 2004, in press.
S53
[26] Chetrite GS, Kloosterboer HJ, Pasqualini JR. Effect of tibolone (Org OD14) and its metabolites on estrone sulphatase activity in MCF-7 and T-47D mammary cancer cells. Anticancer Res 1997;17:135–40. [27] Selcer KW, Li P-K. Estrogenicity, antiestrogenecity and estrone sulfatase inhibition of estrone-3-amine and estrone3-thiol. J Steroid Biochem Mol Biol 1995;52:281–6. [28] Howarth NM, Purohit A, Reed MJ, Potter BVL. Estrone sulfamates: potent inhibitors of estrone sulfatase with therapeutic potential. J Med Chem 1994;37:219–21. [29] Purohit A, Williams GJ, Roberts CJ, Potter BVL, Reed MJ. In vivo inhibition of oestrone sulphatase and dehydroepiandrosterone sulphatase by oestrone-3-O-sulphamate. Int J Cancer 1995;63:106–11. [30] Elger W, Schwarz S, Hedden A, Reddersen G, Schneider B. Sulfamates of various estrogens are prodrugs with increased systemic and reduced hepatic estrogenicity at oral application. J Steroid Biochem Mol Biol 1995;55:395– 403. [31] Valigora SD, Li P-K, Dunphy G, Turner M, Ely DL. Steroid sulfatase inhibitor alters blood pressure and steroid profiles in hypertensive rats. J Steroid Biochem Mol Biol 2000;73:113– 22. [32] Anderson CJ, Lucas LJH, Widlanski TS. Molecular recognition in biological systems: phosphate esters vs. sulfate esters and the mechanism of action of steroid sulfatase. J Am Chem Soc 1995;117:3889–90. [33] Boivin RP, Luu-The V, Lachance R, Labrie F, Poirier D. Structure-activity relationships of 17alpha-derivates of estradiol as inhibitors of steroid-sulfatase. J Med Chem 2000;43:4465–78. [34] Poirier D, Boivin RP. 17␣-Alkyl- or 17␣-substituted benzyl17-estradiols: a new family of estrone-sulfatase inhibitors. Bioorg Med Chem Lett 1998;8:1891–6. [35] Ciobanu LC, Boivin RP, Luu-The V, Labrie F, Poirier D. Potent inhibition of steroid sulfatase activity by 3-O-sulfamate 17␣-benzyl (or 4-tert-butylbenzyl)estra-1,3,5(10)-trienes: combination of two substituents at positions C3 and C17␣ of estradiol. J Med Chem 1999;42:2280–6. [36] Purohit A, Vernon KA, Hummelinck AEW, Woo LW, Hejaz HA, Potter BV, Reed MJ. The development of a-ring modified analogues of oestrone-3-O-sulphamate as potent steroid sulphatase inhibitors with reduce oestrogenecity. J Steroid Biochem Mol Biol 1998;64:269–76. [37] Purohit A, Woo LWL, Barrow D, Hejaz HA, Nicholson RI, Potter BV, Reed MJ. Non-steroidal and steroidal sulfamates: new drugs for cancer therapy. Mol Cell Endocr 2001;171:129– 35. [38] Malini B, Purohit A, Ganeshapillai D, Woo LWL, Potter BVL, Reed MJ. Inhibition of steroid sulphatase activity by tricyclic coumarin sulphamates. J Steroid Biochem Mol Biol 2000;75:253–8. [39] Woo LWL, Lightowler M, Purohit A, Reed MJ, Potter BVL. Heteroatom-substituted analogues of the active-site directed inhibitor estra-1,3,5(10)-trien-17-one-3-sulphamate inhibit estrone sulphatase by a different mechanism. J Steroid Biochem Mol Biol 1996;57:79–88.
S54
J.R. Pasqualini / Maturitas 46S1 (2003) S45–S54
[40] Woo LWL, Purohit A, Malini B, Reed MJ, Potter BVL. Potent active site-directed inhibition of steroid sulphatase by tricyclic coumarin-based sulphamates. Chem Biol 2000;7:773– 91. [41] Pasqualini JR, Chetrite G. Paradoxical effect of estradiol: it can block its own bioformation in human breast cancer cells. J Steroid Biochem Mol Biol 2001;78:21–4. [42] Pollow K, Boquoi E, Baumann J, Schmidt-Gollwitzer M, Pollow B. Comparison of the in vitro conversion of estradiol-17 to estrone of normal and neoplastic human breast tissue. Mol Cell Endocr 1977;6:333–48. [43] Miettinen M, Mustonen M, Poutanen M, Isomaa V, Wickman M, Soderqvist G, Vihko R, Vihko P. 17-hydroxysteroid dehydrogenases in normal human mammary epithelial cells and breast tissue. Breast Cancer Res Treat 1999;57:175–82. [44] Poutanen M, Moncharmont B, Vihko R. 17-Hydroxysteroid dehydrogenase gene expression in human breast cancer cells: regulation of expression by a progestin. Cancer Res 1992;52:290–4. [45] Bonney RC, Reed MJ, Davidson K, Beranek PA, James VHT. The relationship between 17-hydroxysteroid dehydrogenase activity and oestrogen concentrations in human breast tumours and in normal breast tissue. Clin Endocr 1983;19:727–39. [46] Couture P, Theriault C, Simard J, Labrie F. Androgen receptor-mediated stimulation of 17-hydroxysteroid dehydrogenase activity by dihydrotestosterone and medroxyprogesterone acetate in ZR-75-1 human breast cancer cells. Endocrinology 1993;132:179–85. [47] Nguyen B-L, Chetrite G, Pasqualini JR. Transformation of estrone and estradiol in hormone-dependent and hormoneindependent human breast cancer cells. Breast Cancer Res Treat 1995;34:139–46. [48] Peltoketo H, Isomaa VV, Poutanen MH, Vihko R. Expression and regulation of 17-hydroxysteroid dehydrogenase type 1. J Endocrinol 1996;150:S21–30. [49] Prudhomme JF, Malet C, Gompel A, Lalardie JP, Ochoa C, Boue A, Mauvais-Jarvis P, Kuttenn F. 17-Hydroxysteroid dehydrogenase activity in human breast epithelial cells and fibroblast cultures. Endocrinology 1984;114:1483–9.
[50] Gompel A, Malet C, Spritzer P, Lalardrie J-P, Kuttenn F, Mauvais-Jarvis P. Progestin effect on cell proliferation and 17-hydroxysteroid dehydrogenase activity in normal human breast cells in culture. J Clin Endocrinol Metab 1986;63:1174–80. [51] Coldham NG, James VHT. A possible mechanism for increased breast cell proliferation by progestins through increased reductive 17-hydroxysteroid dehydrogenase activity. Int J Cancer 1990;45:174–8. [52] Adams EF, Coldham NG, James VHT. Steroidal regulation of oestradiol-17-dehydrogenase activity of the human breast cancer cell line MCF-7. J Endocrinol 1988;118:149–54. [53] McNeill JM, Reed MJ, Beranek PA, Newton CJ, Ghilchik MW, James VHT. The effect of epidermal growth factor, transforming growth factor and breast tumour homogenates on the activity of oestradiol 17-hydroxysteroid dehydrogenase in cultured adipose tissue. Cancer Lett 1986;31:213–9. [54] Malet C, Vacca A, Kuttenn F, Mauvais-Jarvis P. 17-Estradiol dehydrogenase (E2DH) activity in T47D cells. J Steroid Biochem Mol Biol 1991;39:769–75. [55] Chetrite GS, Ebert C, Wright F, Philippe J-C, Pasqualini JR. Effect of medrogestone on 17-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. J Steroid Biochem Mol Biol 1999;68:51–6. [56] Qian Y, Deng C, Song W-C. Expression of estrogen sulfotransferase in MCF-7 cells by cDNA transfection suppresses the estrogen response: potential role of the enzyme in regulating estrogen-dependent growth of breast epithelial cells. J Pharmacol Exp Ther 1988;2:555–60. [57] Falany JL, Falany CN. Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines. Cancer Res 1996;56:1551–5. [58] Falany JL, Falany CN. Regulation of estrogen activity by sulfation in human MCF-7 human breast cancer cells. Oncol Res 1997;9:589–96. [59] Chetrite GS, Pasqualini JR. The selective estrogen enzyme modulator (SEEM) in breast cancer. J Steroid Biochem Mol Biol 2001;76:95–104.