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PubMed1993

Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures.

Estradiol levels in breast tumors from post-menopausal women are similar to those in pre-menopausal women even though plasma estrogens are much lower after the menopause. In situ estrogen production by the tumor provides a potential means of maintaining high estradiol levels in post-menopausal breast cancer tissue. The estrone sulfatase pathway has been proposed as the mediator of in situ estrogen production. A number of studies suggest that estrone sulfate may be converted into estradiol in breast tumors via the catalytic activity of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase. However, these studies used pharmacologic levels of estrogen sulfates and have not shown that physiologic levels can support biologic effects. Accordingly, the present study examined the dose relationship of estrone sulfate to a variety of biologic endpoints in MCF-7 breast cancer cells in culture. These cells converted physiologic concentrations of estrone sulfate to quantities of free estradiol capable of stimulating cell growth. Under these conditions, the nuclear steroids observed were free estrone and estradiol. Increase in cell number after 6 days of exposure to steroid required 100 nM estrone sulfate. However, S-phase, a more sensitive measure of cell proliferation, was stimulated by 0.1 nM estrone sulfate, a clearly physiologic concentration. Stimulation of estrogen-dependent protein markers such as pS2 and progesterone receptor required much higher concentrations of estrone sulfate. These effects were mediated through the estrogen receptor since the pure anti-estrogen, ICI 164384, blocked all effects produced by estrone sulfate. While it has been suggested that anti-estrogens may partly exert their effects by inhibition of sulfatase and 17 beta-hydroxysteroid dehydrogenase, this did not occur under our experimental conditions. These data provide evidence of the relevance of the estrone sulfatase pathway since biologic effects can be demonstrated in response to physiologic concentrations of estrone sulfate.

Biological Transport↗

PubMed2005

Suppression of cell proliferation by inhibition of estrone-3-sulfate transporter in estrogen-dependent breast cancer cells.

PURPOSE: The aim of the study is to suppress the progress of estrogen-dependent breast cancer by inhibiting the membrane transporter, which mediates the internalization of estrone-3-sulfate as estrogen precursor in the cancer cells. METHODS: The uptake of estrone-3-sulfate by estrogen-dependent breast cancer MCF-7 cells was measured, and inhibitory study using various organic anions on estrone-3-sulfate uptake by MCF-7 cells was conducted. The effects of the inhibitor on the transcription of reporter gene and cell proliferation induced by estrone-3-sulfate were examined. RESULTS: The uptake of estrone-3-sulfate by MCF-7 cells was saturable with Km value of 2.32 microM. The uptake was Na+-independent and was inhibited by several anionic compounds such as bromosulfophthalein. Bromosulfophthalein also significantly inhibited the transcription of reporter gene via estrogen response element and cell proliferation induced by estrone-3-sulfate. However, the transcriptional activation or cell proliferation induced by estrone was not inhibited by bromosulfophthalein. Reverse transcription-polymerase chain reaction analysis revealed the expression of mRNA of organic anion transporting polypeptide (OATP)-D and OATP-E as possible candidates to transport estrone-3-sulfate. CONCLUSIONS: The uptake of estrone-3-sulfate is mediated by Na+-independent transporter(s). Inhibitor of estrone-3-sulfate transporter suppressed the transcription and cell proliferation induced by estrone-3-sulfate in MCF-7 cells. The results provide the basis of a novel strategy for breast cancer treatment by focusing on the transporter responsible for the uptake of estrone-3-sulfate.

Alkaline Phosphatase↗

PubMed2002

Peripheral changes in estrone sulfate concentration during the first trimester of gestation in cattle: comparison with unconjugated estrogens and relationship to fetal number.

Estrone sulfate originates mainly in the conceptus during gestation in cattle. Its concentration in maternal body fluids is a useful indicator of placental function. The objective of this study was to determine the profiles of estrone sulfate during early gestation in singleton and twin bearing cows using a newly developed extraction method. One or two blastocysts produced in vitro were nonsurgically transferred to regularly cycling Holstein cows on Day 7 (Day 0 was defined as the first day of standing estrus). Pregnancy was diagnosed on Days 30, 45 and 60 by transrectal ultrasonography and finally confirmed at parturition. Six cows with singleton and six with twin pregnancies were used in the experiment. Blood was collected every other morning by jugular venipuncture from the day after transfer to Day 100. Harvested plasma was applied to reversed-phase C18 cartridges. Estrone sulfate and unconjugated estrogens (estrone and estradiol-17beta) retained in the cartridge were eluted separately by methanol stepwise gradient and each measured by validated radioimmunoassay. On average, estrone sulfate concentrations fluctuated between 2 and 6 pg/ml until Day 50 in both groups and then gradually increased. However, the levels of estrone and estradiol- 17beta remained low (1-5 pg/ml) until Day 80. The concentration of estrone sulfate after Day 50 was significantly affected by the day of gestation (P < 0.0001) and the number of fetuses (P < 0.01). After Day 80. estrone sulfate increased drastically, followed by increases in estrone and estradiol-17beta concentrations. The rate of increase in estrone sulfate during Days 80-100 was the greatest among all estrogens (P < 0.05). The rates of increase in estrone sulfate during Days 50-80 and 80-100 were 1.7 times greater in twin pregnancies than in cows having one fetus. These results suggest that the concentration of estrone sulfate in bovine peripheral blood plasma during early gestation has potential application in monitoring embryonic growth as well as fetoplacental development.

Animals↗

PubMed1994

Conversion of estrone to 2- and 4-hydroxyestrone by hamster kidney and liver microsomes: implications for the mechanism of estrogen-induced carcinogenesis.

As part of an ongoing investigation of the role of metabolic activation of estrogens in the genesis of cancers such as estrogen-induced renal tumors in hamsters, we have 1) determined steroid-17 beta-oxidoreductase activity of microsomes and cytosol prepared from hamster kidney and liver; 2) compared the rates of 2-, 4-, and 16 alpha-hydroxylations of estrone by microsomes from hamster kidney and liver; and 3) determined the rates of inactivation of 2- and 4-hydroxyestrone by catechol-O-methyltransferase from hamster kidney and by purified enzyme. Microsomal steroid-17 beta-oxidoreductase activity in hamster kidney and liver was low and favored the conversion of estrone to estradiol. Cytosolic steroid-17 beta-oxidoreductase activity was only barely detectable in both liver and kidney. Using hepatic microsomes, the rate of 2-hydroxylation of estrone was comparable to that found previously using estradiol as substrate, whereas 4-hydroxylation of estrone was double that of estradiol. Using renal microsomes, the rates of 2- and 4-hydroxylation of estrone were 10- to 20-fold higher than those with estradiol as substrate, and the ratio of 2- to 4-hydroxylation was about 2:1. Fadrozole hydrochloride was an equally good inhibitor of rates of 2- and 4-hydroxylation of estrone (20 microM) by hepatic microsomes (IC50, approximately 25 microM). Corresponding IC50 values with renal microsomes were less than 2 microM, and 2-hydroxylation of estrone was inhibited by Fadrozole hydrochloride up to 15% more than 4-hydroxylation. Treatment of hamsters with estradiol for 2 months decreased rates of 2- and 4-hydroxylation of estrone by renal microsomes by approximately 95%. The rate of conversion of estrone to 16 alpha-hydroxyestrone by hepatic microsomes was 10-20% that of 2-hydroxylation. Renal microsomes catalyzed 16 alpha-hydroxylation of estrone at an even lower rate (approximately 5% of that of 2-hydroxylation). Rates of O-methylation of 2- and 4-hydroxyestrone by hamster kidney cytosol were comparable to those of 2- and 4-hydroxyestradiol. In conclusion, conversion of estrone to its catechol metabolites by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, is quantitatively more important than the conversion to 16 alpha-hydroxyestrone. The findings are consistent with the postulated role of catechol estrogens generated in situ in estrone-induced carcinogenesis.

Animals↗

PubMed1981

Radioimmunoassay of plasma equilin and estrone in postmenopausal women after the administration of premarin.

A RIA for the measurement of plasma equilin (3-hydroxy-1,3,5-(10)7-estratetraen-17-one) and estrone in postmenopausal women and other estrogen-deficient women on exogenous equine estrogen replacement therapy is described. Antiserum against estriol-3,16,17-trihemisuccinate-HSA and high specific activity [2,4-3H]equilin and [6,7-3H]estrone were used in the assay procedure. Specificity of the assay was achieved by separation of equilin from estrone by chromatography on micro-Celite partition columns using silver nitrate as the stationary phase. The sensitivity of the standard curves for both equilin and estrone was 10-20 pg, and the smallest amount of estrone and equilin that could be measured accurately in the plasma was 250 pg/ml for both steroids. The coefficient of variation for both steroids ranged from 1-8%, over a range of 250-5000 pg/ml plasma. The interassay coefficients of variation for equilin and estrone were 4.6% and 2.4%, respectively. After the administration of 10 mg Premarin iv, maximum concentrations of 4 and 11.2 ng/ml for equilin and estrone, respectively, were obtained after 20 min. Thereafter, both steroids disappeared from the plasma gradually. When 10 mg Premarin were administered orally, equilin and estrone appeared in the blood gradually, and maximum levels of 560 and 1400 pg/ml were reached after 3 and 5 h for equilin and estrone, respectively. Equilin gradually disappeared, and by 24 h, only small amounts (125 pg/ml) were detectable. The levels of estrone declined more rapidly, though it was still detectable after 24 h. These preliminary results indicate that equilin sulfate is converted to circulating unconjugated equilin in a manner similar to the conversion of circulating estrone sulfate to estrone.

17-Ketosteroids↗

PubMed1983

Interaction between rat alpha 1 fetoprotein and fluorescent derivatives of estrone in relation to the position and type of the fluorescent label.

A series of derivatives of estrone with fluorescent dyes (dansyl or coumarin) coupled at different positions on the steroid molecule, have been synthesized. These derivatives were tested for their quantum yields and their binding properties were determined with respect to rat alpha 1 fetoprotein. Derivatives at C-3 (estrone-3-hemisuccinate-dansyl-cadaverine and estrone-3-dansyl) compete with estrone for binding to the fetal protein; however derivates of estrone at C-6 (estrone-6-carboxymethyloxime-dansyl-cadaverine) and at C-17 (estrone-17-carboxymethyloxime-coumarine, estrone-17-carboxymethyloxime-dansyl-cadaverine and estrone-17-dansyl-hydrazine) compete poorly or not at all. The association constant of the radioactive derivative estrone-3-dansyl [3H] with rat alpha 1 fetoprotein was measured directly: the same number of high affinity binding sites (0.6) as that for estrone was found with an apparent association constant of 3.7 X 10(6) M-1. In addition to the high affinity binding sites, a low affinity class of binding sites was found which corresponds to the binding of the dansyl fraction of the fluorescent steroid derivative.

Animals↗

PubMed1995

Estrone-induced cell proliferation and differentiation in the mammary gland of the female Noble rat.

In this study we examined the influence of estrone on proliferation and differentiation in the mammary gland of female Noble rats. Estrone treatment increased proliferation in mammary epithelial cells at days 3 and 7, and peaked by day 11 of estrone exposure. In addition, estrone exposure altered cell cycle kinetics. Animals exposed to estrone for 11 days demonstrated a 9-fold increase in the cells in G1 phase and a 48-fold increase in the cells in S phase, compared to those of controls. Differentiation, measured by the degree of lobular maturation, was significantly increased in the treatment group by day 7 and continued to mature through day 21 post estrone implant. The appearance of the mammary glands in these animals approached the morphology normally found during pregnancy and lactation. In a subset of animals we examined the effects of a concurrent treatment with luteolin, a bioflavonoid which has been shown to inhibit estrone binding to type II estrogen receptors. Luteolin significantly reversed the effects of estrone treatment on both proliferation and differentiation in these animals. Changes in proliferation and cell cycle kinetics have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in proliferative cells by > 2-fold and a perturbation in cell cycle kinetics from estrone exposure. These changes may play a role in the induction of estrone-induced mammary cancer in the Noble rat model. In addition, the anti-proliferative action of luteolin suggests that it may play a protective role in estrone-induced mammary carcinogenesis.

Animals↗

PubMed1996

The effect of estrone and estradiol treatment on endometrial total protein, uteroferrin, and retinol-binding protein secretion during midpregnancy or midpseudopregnancy in swine.

It has been hypothesized that conceptus estrogens influence endometrial protein secretion during pregnancy in swine. To test this hypothesis, the effect of estrone and estradiol treatment from d 30 to 60 of pregnancy or pseudopregnancy on endometrial protein secretion was investigated. Pregnant (P; n = 16) and pseudopregnant (PP) gilts (n = 18) received either sham treatment or estrone or estradiol implants (5 mg/d release rate; 60 d release) on d 30 of P or PP. Blood samples were collected on d 30, 40, 50, and 60 to measure estrone and estradiol. On d 60, gilts were hysterectomized. For P gilts, endometrium in apposition to one placenta from each uterine horn was collected. For PP gilts, each uterine horn was flushed with 40 mL of leucine-deficient minimal essential medium (MEM), and endometrial tissue was collected from each horn. Endometrial tissues were incubated in MEM in the presence of 50 microCi of [3H]leucine to examine protein secretion. Estrone and estradiol treatments increased both plasma and endometrial concentrations of estrone (P < .01 except endometrium for P gilts) and estradiol (P < .01, respectively). Endometrium from P gilts secreted more nondialyzable macromolecules (NDM), acid phosphatase activity (AP, a measure of uteroferrin), and retinol-binding protein (RBP) in culture than did endometrium from PP gilts. Estrone treatment increased (P < .01) endometrial NDM from P gilts but not that from PP gilts; estradiol had no effect. Both estrone and estradiol increased (P = .069) endometrial secretion of AP of PP but not of P gilts. Endometrial secretion of RBP was not affected by either estrone or estradiol treatment. Neither estrone nor estradiol affected total protein or AP and estrone treatment decreased (P < .05) RBP in uterine flushings from PP gilts. These data indicate that endometrium from P pigs secretes more protein than endometrium from PP pigs but neither estrone nor estradiol completely mimics the effect of pregnancy.

Acid Phosphatase↗

PubMed1986

Estrone sulfate: a potential source of estradiol in human breast cancer tissues.

Local formation of estradiol in human breast tumors could provide a more important source of estrogen than is delivered from plasma. Prior studies have suggested that estrone is primarily synthesized from estrone sulfate. The enzyme 17 beta-hydroxysteroid dehydrogenase (HSD) would be required to convert estrone to estradiol. This study characterized HSD in 1000 X g supernatants from human breast tumors. Estradiol synthesis was linearly related to tissue concentration or time over the range studied. Cofactor requirements varied with estrone concentration. High and low affinity sites were found in 50% of tissues studied, while the remainder contained only low affinity sites. Screen assays showed measurable activity in all 42 samples tested. This activity ranged from 0.73- greater than 100 nmol estrone synthesized/g protein/hr, with a median activity of 5.9 nmol/g/hr. We evaluated the biological relevance of the sulfatase-HSD pathway by testing the ability of estrone sulfate to stimulate colony formation in soft agar cultures of nitrosomethylurea-induced rat mammary tumors. The maximally effective concentration ranged from 10(-7) to 10(-4)M. Significant stimulation of colony formation was observed in 7 of 8 experiments. The estrone sulfate stimulation pattern was similar to that previously observed with estradiol. Of the 3H-estrone sulfate added to the dishes, 20-98% was recovered as estrone and 0.2-6% as estradiol. These studies suggest that the requisite enzymes are present in human breast tumors for conversion of estrone sulfate to estradiol, and that this pathway may be biologically significant.

17-Hydroxysteroid Dehydrogenases↗

PubMed1979

Antisera reactive directly to estrone sulfate.

Derivatives of estrone were prepared and linked to bovine serum albumin or its methyl-esterified form to produce immunogens which were effective in raising antisera to estrone sulfate. The most effective was estrone-3-methylphosphonothioate, electrostatically complexed with methylated bovine serum albumin. The ionically combined hapten functioned as an antigenic determinant as do covalently bound haptens when administered to sheep in emulsions with Freund's complete adjuvant. Estrone-3-phosphate covalently or electrostatically linked to bovine serum albumin also produced antisera reactive to estrone sulfate. Estrone sulfate itself, after electrostatically complexing to methylated bovine serum albumin and administration with Freund's complete adjuvant to sheep, was ineffective in producing antisera. The sera which had workable titres to estrone sulfate showed considerable cross-reaction with free estrone but was otherwise highly specific with little or no reaction with other steroid sulfates, glucosiduronates or other free steroids. Radioimmunoassay curves using [6,7-3H]-estrone sulfate were highly sensitive and were effective in the range of 5-250 pg estrone sulfate.

Cross Reactions↗

PubMed1998

Pharmacokinetics and pharmacodynamics of a novel estrogen delta8-estrone in postmenopausal women and men.

Recently a tenth equine estrogen, identified as the sulfate ester of delta8-estrone has been reported to be present in Premarin (a conjugated equine estrogen preparation), and because of its unique ring B unsaturated structure (conjugated double bond in the B ring), we have, in the present study, determined its pharmacokinetics in postmenopausal women and men, its interaction with uterine estrogen receptors and its uterotropic activity. After the administration of [14C]delta8-estrone, blood was drawn at various time intervals, and the plasma fractionated into the unconjugated sulfate and glucuronide fractions. The disappearance of radioactivity as delta8-estrone from plasma can be described as a function of two exponentials. The half-lives of the first and second components were 5+/-0.2 and 40.4 min, respectively. The mean metabolic clearance rate calculated (MCR), was 1711+/-252 l/d m2. From the unconjugated fraction, delta8-17beta-estradiol was also isolated and identified. From the sulfate conjugated fraction, delta8-estrone sulfate and delta8-17beta-estradiol sulfate were isolated in almost equal amounts. No other metabolites of delta8-estrone was detectable in the plasma. Both delta8-estrone and delta8-17beta-estradiol bind with human endometrial and rat uterine estrogen receptors with high affinity. The binding affinities of delta8-17beta-estradiol for human endometrial and rat uterine cytoplasmic receptors were 4 and 25 times higher than those of the parent estrogen delta8-estrone, respectively. Administration of delta8-estrone and delta8-17beta-estradiol (2 microg/100 g body weight) to immature rats significantly (P< 0.05) increased the uterine weight compared to the controls. These data demonstrate that delta8-estrone has estrogenic activity, and that it is further metabolized in man to a single more potent estrogen, delta8-17beta-estradiol. The extent of this activation by 17beta-reduction appears to be greater than that observed with other estrogens. Both estrogens circulate as sulfate conjugates and are very slowly eliminated from the circulation. These data further suggest that delta8-estrone and its major metabolite delta8-17beta-estradiol can contribute to the overall in vivo biological effects of Premarin.

Adult↗

PubMed1999

Short-term treatment with estrone oleate in liposomes (Merlin-2) does not affect the expression of the ob gene in Zucker obese rats.

Young female Zucker fa/fa rats of 370-430 g were implanted with osmotic minipumps releasing 3.5 micromol/day-kg of estrone oleate in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a sustained loss of appetite, and a decrease in body weight of 3.5%, which contrasts with the 8.2% increase in controls during the period studied. Plasma insulin, glucose and urea decreased, and liver glycogen increased with Merlin-2 treatment. Plasma ACTH and corticosterone increased to a maximum at the end of the experiment. The expression of the ob gene in adipose tissue was unchanged, and plasma leptin levels were also unchanged by treatment. Estrone levels increased more than 1500-fold, and estrone oleate rose 100-fold during treatment. The fact that estrone oleate had no effect on the leptin levels or expression in obese rats, in contrast with the marked inhibition observed in the lean suggests that the functionality of the leptin receptor is essential for estrone oleate inhibition of the ob gene. This also suggests that leptin may control ob gene expression in white adipose tissue and that estrone oleate may activate this process. The slimming effect of estrone oleate is, thus, not directly dependent on leptin, since both normoleptinemic and hyperleptinemic animals lose fat following treatment nor are the effects on appetite and energy expenditure mediated by leptin. However, leptin levels and the expression of the ob gene are directly linked with estrone oleate function. A possible involvement of leptin in estrone oleate action is postulated. The results support the participation of estrone oleate in the control of body weight and hint at the complexity of its regulation by leptin and glucocorticoids.

Animals↗

PubMed2006

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1).

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.

Binding Sites↗

PubMed1979

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine.

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.

Journal Article↗

PubMed1993

Characterization of estrone sulfatase activity in human thrombocytes.

Estrone sulfatase is an important enzyme which catalyzes the production of estrone from estrone sulfate in a variety of human and animal tissues. We report, for the first time, on the presence of estrone sulfatase activity in thrombocytes from human blood. Incubation of [3H]estrone sulfate in the presence of human thrombocyte lysates resulted in the formation of [3H]estrone as assessed by two-dimensional TLC. Estrone sulfatase activity was localized in the mitochondrial-microsomal fraction in thrombocytes from human blood. The enzyme was thermostable and had an optimum pH of 5.60 in acetate buffer. The highest activity was obtained in the presence of 0.1% of either Nonidet P-40 or Triton X-100. Phosphate ions (1 mM) inhibited the enzyme activity by 64% and similar effects were observed in the presence of platelet-free plasma. Endogenous inhibitors had no effect on the observed enzyme activity under assay conditions as evidenced in this study. The apparent Km value was 3.16 +/- 0.08 microM for [3H]estrone sulfate and V was 188.5 +/- 2.6 (mean +/- SEM, n = 22) pmol.mg protein-1.h-1. Comparison between two thrombocyte preparative procedures provided evidence that thrombocyte estrone sulfatase activity should be measured in thrombocyte samples representing the whole thrombocyte population. This parameter appeared critical for accurate measurements of enzyme activity. The presence of estrone sulfatase activity in human thrombocytes provides a new non-invasive tool for the study of this activity both in physiological and pathological conditions which could be of potential clinical relevance.

Adult↗

PubMed1996

Synthesis and sulfatase inhibitory activities of non-steroidal estrone sulfatase inhibitors.

About one-third of breast cancers are classified as estrogen-dependent breast cancers. In the past 10 years, numerous reports have suggested the importance of estrone sulfate and estrone sulfatase in regulating the supply of estrogens to these cancers. Estrone sulfatase inhibitors may thus prove to be useful for the treatment of these diseases. Several research groups have reported the development of estrone sulfatase inhibitors, and estrone-3-O-sulfamate has been shown to be the most potent sulfatase inhibitor. However, a recent report indicated that estrone may be released during the inactivation of sulfatase by estrone-3-O-sulfamate and rendered the inhibitor to be estrogenic. Therefore, there is a need for a potent non-steroidal sulfatase inhibitor that is metabolically stable, more selective, and lacking estrogenic activity. We developed a series of (p-O-sulfamoyl)-N-alkanoyl tyramines, and they proved to be potent estrone sulfatase inhibitors. Using human placental microsome as the enzyme source, the best inhibitor in this series, compound 18, has an IC50 of 55.8 nM. Another potent inhibitor in this series, compound 17, exhibited time-dependent inactivation of sulfatase when incubated at various concentrations (0.2-1.0 microM) of the inhibitor. Estrone sulfate partially blocked the inactivation of the enzyme by the compound, indicating that the compound inactivated sulfatase at the active site. The irreversible nature of the enzyme-inhibitor interaction was supported by irreversibility studies. Thus, (p-O-sulfamoyl)-N-alkanoyl tyramines represent a new series of non-steroidal estrone sulfatase inhibitor.

Binding Sites↗

PubMed1984

In situ estrogen production via the estrone sulfatase pathway in breast tumors: relative importance versus the aromatase pathway.

Estrone and estradiol concentrations in breast tumor tissue are an order of magnitude higher than circulating plasma levels in postmenopausal women with breast cancer. Local production of estrogen in the neoplastic tissue is one of several possible explanations for this plasma/tissue gradient. This study evaluated breast tumor estrogen production via the estrone sulfate to estrone (sulfatase) pathway and compared this with the androstenedione to estrone (aromatase) system in human and rodent mammary tumors. Estrogen production from estrone sulfate was related linearly with time and tissue concentrations, exhibited an apparent Km of 20 microM, and produced a linear Eadie-Hofstee kinetic plot consistent with a single class of enzymatic sites. Measurement of sulfatase in 35 human breast tumors using enzyme saturating conditions revealed estrone production ranging from 0.8-125 mumol/g protein . h. The corresponding range in host mammary tumors was 3.5-7.1 mumol/g protein . h. In human breast tumors, sulfatase activity did not correlate with the levels of estrogen receptor or progesterone receptor. Comparison of sulfatase with aromatase activity in human tumors at physiological levels of substrate revealed estrone formation via sulfatase of 2.8 pmol estrone produced/g protein . h, while aromatase produced only 0.27 pmol/g protein . h. In rat mammary tumors, sulfatase activity was similar to that in human tumors, whereas aromatase activity could not be detected, even with a highly sensitive assay. Thus, estrone sulfatase appears to be the enzyme primarily responsible for intratissue estrone production in hormone-dependent breast carcinomas.

Animals↗

PubMed1982

Interconversion of estrone and estradiol-17 beta in lung slices of the adult human.

The metabolism of tritium-labeled estrone and estradiol-17 beta in slices of lung tissue obtained from an adult human was studied; estrone was identified as the only metabolite of estradiol-17 beta and estradiol-17 beta as the exclusive product of estrone metabolism. Product formation remained linear as a function of time of incubation up to 3 h and of wet lung tissue mass up to 300 mg/ml. At equimolar substrate concentrations, the rates of estrone formation were at least 2-fold greater than those of estradiol-17 beta. The apparent KM of 17 beta-hydroxysteroid oxidoreductase for estrone was 11 microM and that for estradiol-17 beta was 10 microM. These results are suggestive that the human lung enzyme binds estrone and estradiol-17 beta with similar affinities; however, the oxidative pathway is favored as indicated by the greater Vmax attained in the formation of estrone. It is possible that, in vivo, the human lung constitutes a site for estradiol-17 beta inactivation to estrone as well as a site for the conversion of estrone to estradiol-17 beta. This last process may become particularly important in instances in which the ovaries have ceased to function and secrete estradiol-17 beta, e.g. the postmenopausal women.

Carcinoma, Squamous Cell↗