Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC₅₀ below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC₅₀ below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 μM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.     

The role of estrogens on the proliferation of human breast tumor cells (MCF-7)

The cloned human breast tumor cell line C7MCF7-173 behaved as an estrogen-dependent tumor in the nude mice. In contrast, E2 added to serum-less media did not increase the multiplication rate of these cells over the values obtained in the control cultures. Media supplemented with charcoal-dextran stripped (CD) human female serum (FHS) resulted in inhibition of cell proliferation in a concentration-dependent pattern (40% = 20% greater than 10% greater than 5% greater than 2.5%). E2 addition to all but the 2.5% CDFHS significantly increased the proliferation rate of these cells. The E2 concentration required to attain maximal proliferation rate increased as the serum concentration of the medium increased (e.g. 3 X 10(-11)M for 10% CDFHS, 3 X 10(-10)M for 40% CDFHS). E2 concentrations higher than the one needed to achieve maximal proliferation rate resulted in decreased cell yields (shut-off mechanism). Similar effects were obtained with synthetic and other natural estrogens. CD fetal bovine serum (FBS) also inhibited the proliferation of C7MCF7-173 cells; however, at similar concentration the inhibitory effect of CDFHS was more potent than the one obtained with CDFBS. The addition of "growth factors" (insulin, Epidermal Growth Factor and transferrin) and non-estrogenic steroids to 10% CDFHS failed to overcome the inhibitory effect of this serum. These results suggest that: (1) human and fetal bovine sera contain a specific inhibitor of the proliferation of E2-sensitive cells (estrocolyones), and (2) E2 promotes cell proliferation by neutralizing this inhibitor.     

Developmental patterns of plasma dehydroepiandrosterone sulfate and testosterone in male pigs

The relationship between plasma levels of dehydroepiandrosterone sulfate (DHAS) and testosterone (T) was determined by radioimmunoassays in growing and adult pigs. Seven young males were bled at 2-weekly intervals between 1 and 47 weeks of age and two adult boars were cannulated for short-term studies. Plasma samples were extracted with methylene chloride and T was isolated by Celite chromatography. DHAS was assayed directly in the aqueous phase.Dehydroepiandrosterone occurred predominantly (89.7 ± 10.6%) as the sulfoconjugate in boar plasma (n = 50). Plasma DHAS was undetectable in castrated males (n = 2). At 1 week of age, mean levels (± S.D.) of DHAS and T were 5.0 ± 3.0 ng/ml and 0.15 ± 0.10 ng/ml, respectively; and they rose to small peaks of 16.0 ± 2.0 ng/ml and 0.63 ± 0.10 ng/ml at 3 weeks. At 7 weeks, the levels of DHAS and T increased gradually from 10.0 ± 6.7 and 0.11 ± 0.10 ng/ml to 27.0 ± 6.6 and 1.84 ± 0.61 ng/ml at 19 weeks. There followed a marked increase to 4.90 ± 3.30 ng/ml at 21 weeks for T and a less abrupt rise to 44.0 ± 9.3 ng/ml at 23 weeks for DHAS. The mean levels remained high from then onwards, fluctuating between 24.0 ± 8.7 and 54.5 ± 5.0 ng/ml for DHAS and between 1.73 ± 0.86 and 4.43 ± 1.26 ng/ml for T. Episodic fluctuations were noted in two boars during hourly collection for 24 h, with mean levels of 9.0 ± 4.9 and 50.0 ± 10.4 ng/ml for DHAS, and 1.76 ± 0.83 and 3.26 ± 0.63 ng/ml for T, respectively.For all ages of males, plasma DHAS and T levels were highly correlated (r = 0.95) with greater concentrations of DHAS in all samples. Although individual differences in steroid profiles were noted, concentrations for DHAS and T showed almost parallel increases at puberty and corresponding fluctuations in adult boars. It is suggested that plasma DHAS determinations provide a simple, sensitive assessment of androgen production in the male pig.     

Antisense-mediated Inhibition of the plasma membrane calcium-ATPase suppresses proliferation of MCF-7 cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.     

Quantitative analysis of estrogens and estrogen metabolites in endogenous MCF-7 breast cancer cells by liquid chromatography-tandem mass spectrometry

To study the roles of estrogens and estrogen metabolites (EMs) in breast carcinogenesis, we reported a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing selective reaction mode (SRM) to analyze estrogens and EMs in the extracellular and intracellular compartments of endogenous MCF-7 breast cancer cells through simple ethyl acetate (EA) extraction and dansyl chloride derivatization. Under a 35-min LC gradient elution on a reversed phase C18 column, the method was shown to simultaneously quantify 12 estrogens and EMs: estrone (E1) and its 2-, 4-, 16α-hydroxy derivatives (2-OHE1, 4-OHE1, 16α-OHE1), and 2-, 4-methoxy derivatives (2-MeOE1, 4-MeOE1); 17β-estradiol (E2) and its 2-, 4-hydroxy derivative (2-OHE2, 4-OHE2) and 2- and 4-methoxy derivatives (2-MeOE2 and 4-MeOE2); and estriol (E3), using ethinylestradiol (EE2) as the internal standard (IS). Using a calibration curve-standard addition hybrid method, we were able to determine the amount of estrogens and EMs in not only the treated cells but also the non-treated cells. The limits of quantification (LOQs) were determined to range from 0.05-80 pg on column with an inter-batch accuracy around 72-123% and precision around 1-10%. Results indicated that trace amounts (<0.9 fg/cell) of E1 and E2 were present in both the extra- and intra-cellular compartments under non-treated condition but DMSO could induce E1 and E2 as well as trace amounts (<2.25 fg/cell) of EMs in the cell. E2 treatment substantially increased not only E1 and E2 in the intra-cellular (60 fg/cell) and extra-cellular (3000 fg/cell) compartment but also substantially induced EMs primarily in the extracellular compartment (0.6-25 fg/cell). These data implied that EMs could be quickly generated and distributed to the extracellular compartment by E2 within 24h of treatment and DMSO solvent could potentially induce slight estrogen effects.     

Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells

When MCF-7 cells were incubated with 10 or 20 microM CdCl(2), p53 protein level increased after 18 h. Among serines in p53 protein immunoprecipitated from cells treated with CdCl(2), only Ser 15 was phosphorylated. No clear phosphorylation was found on Ser 6, 9, 20, 37, and 392. Accumulation of p53 protein phosphorylated at Ser 15 was also found after 18 h exposure. While phosphorylation of extracellular signal-regulated protein kinase, c-Jun NH2-terminal kinase and p38 was found in cells treated with CdCl(2), treatment with U0126, LL-Z1640-2, or SB203580 did not suppress Ser 15 phosphorylation. On the other hand, treatment with wortmannin or caffeine suppressed CdCl(2)-induced Ser 15 phosphorylation and accumulation of p53 protein. The present results showed that cadmium induces phosphorylation of p53 at Ser 15 in MCF-7 cells depending on phosphatidylinositol 3-kinase related kinases, but not on mitogen-activated protein kinases.     

Effects of estradiol concentration on levels of nuclear estrogen receptors in MCF-7 breast tumor cells

We have measured the time-course of estrogen receptor levels in nuclei of the estrogen-responsive breast tumor cell line MCF-7 during 90-120 min exposure of the cells to estradiol at physiologic (10(-10)M), pharmacologic (10(-6)M), and an intermediate (10(-8)M) concentration. Cells were preincubated for one week in a serum-free defined medium resembling that of Barnes and Sato, and then incubated in estradiol-containing medium. Nuclei were isolated at various times during the incubation, and filled and unfilled nuclear estrogen receptor levels were assayed. Increasing the concentration of estradiol in the incubation medium from 10(-10)M to 10(-8)M yielded increasing levels of filled nuclear receptor at all times studied, while further increase of the estradiol concentration of 10(-6)M decreased filled receptor levels from 10(-8)M values. Unfilled receptor levels dropped rapidly to zero under 10(-6)M and 10(-8)M estradiol incubation, but remained unchanged under 10(-10)M estradiol incubation. Together these results suggest that high-concentration estradiol may lead to "down-regulation" of filled nuclear receptors, which may be a contributing factor in inhibition of tumor growth. On the other hand, the continued presence of unfilled receptors only under physiological concentrations of estradiol may suggest a role for these receptors in sustaining tumor growth.     

A radioimmunoassay for 7 alpha-hydroxy dehydroepiandrosterone in human plasma.

A radioimmunoassay for plasma 3 beta, 7 alpha-dihydroxy-5-androsten-17-one (7 alpha-hydroxy DHA) has been developed using anti-sera raised against 3 beta, 7 alpha-dihydroxy-5-androstene-17 beta-carboxyl-bovine serum albumin conjugate and [1, 2 (n) - 3H] 7 alpha-hydroxy DHA as the radioligand. Significant cross reactivity was found with 3 beta, 7 alpha-dihydroxy-5-pregnen-20-one (44%), 3 beta, 7 beta-dihydroxy-5-androsten-17-one (6%), 3 beta, 6 beta-dihydroxy-4-androsten-17-one (2.5%), 3 beta-hydroxy-5-androsten-17-one (DHA, 2%), 3 beta, 7 beta-dihydroxy-5-pregnen-20-one (2%) and 7 alpha-hydroxy-4-androstene-3, 20-dione (1%). 7 alpha-Hydroxy DHA was extracted from plasma and separated from cross-reacting factors using alumina micro-columns. The separation of bound and free steroid was achieved using dextran-coated charcoal. The concentration of 7 alpha-hydroxy DHA in the plasma of breast cancer patients was significantly lower than the concentrations in the plasma of normal women, hospitalized women suffering from non-endocrine diseases and patients with benign breast disease. The decrease in the concentration of 7 alpha-hydroxy DHA in the plasma of pregnant women was not significant.     

Reduced conversion of dehydroepiandrosterone into estrogens in women having hypogonadotropic hypogonadism associated with weight loss

The purpose of this study was to evaluate, without using radioisotopes, the peripheral contribution of dehydroepiandrosterone (D) to estrogens and to androstenedione (A) in patients with hypogonadotropic hypogonadism associated with weight loss (HH) and in normal menstruating women (N).Unlabelled D was infused for 48 h in 12 normal women and in 12 women affected by HH. Plasma levels of D, dehydroepiandrosterone sulfate (DS), A, estrone (e₁), estrone sulfate (E_1s) and estradiol (E₂) were measured before and after 48 h of infusion. Metabolic clearance rates of D (MCR_D), production rates of D (PR_D), and increases in plasma concentration of DS, A, E₁, E_1s and E₂, relative to the corresponding increase in plasma concentration of D, were determined. The baseline plasma levels of all steroids studied were found to be significantly lower in the patient group than in the control. The MCR_D in the normal and the HH groups were similar (1420 ± 340 1/day versus 1670 ± 569 1/day, P > 0.05). No significant difference was found in PR_D between the 2 groups (X ± SD 10.3 ± 5 versus 13.3 ± 5.5 mg/day, P > 0.05). Administration of D increased the levels of estrogen in the normal group but not in the HH group. The relative increase in plasma levels of DS resulting from infusion of D (Δ_CDS/Δ_CD) was found to be larger in the HH group than in the normal group (40.4 ± 17 versus 26.3 ± 11.8, P < 0.05). Furthermore, relative increases in plasma levels of A derived from infusion of D were larger in the HH group than in the normal group (0.0495 ± 0.0021 versus 0.192 ± 0.0071, P < 0.001).We conclude from these results that in the HH patients there is a blockage of the peripheral conversion of D to e₁ and E_1s and an enhancement of the peripheral conversions of D to DS and to A. These metabolic changes may account for the androgenization of the patients under study.     

Pendrin transporter carries out iodide uptake into MCF-7 human mammary cancer cells

Previous studies have shown that iodide is actively taken up into mammary alveolar epithelial cells and secreted into milk. In the present studies we demonstrate that 125I also accumulates in MCF-7 cells against a concentration gradient; distribution ratios of greater than 30 were achieved. Iodide uptake into MCF-7 cells is transient, with peak accumulations occurring in about 5 min. The iodide is rapidly metabolized, probably to iodine, and it then exits the cells. The iodide transporter identified in MCF-7 cells is pendrin. DIDS, a nonspecific inhibitor of anion exchange, inhibits iodide uptake. Iodide uptake is impaired at reduced temperature, but is not dependent on sodium. Inhibitors of the sodium-iodide symporter (NIS) as well as ouabain did not affect the extent of iodide uptake. The pendrin transporter but not NIS was identified via western blotting techniques. Pendrin appears to be the primary iodide transporter in the MCF-7 cell line stocks that were employed for these studies.     

Spermatic and peripheral venous plasma concentrations of dehydroepiandrosterone sulfate in prepubertal and pubertal boys

Dehydroepiandrosterone sulfate (DEAS), a major adrenal product, is quantitatively one of the most important steroids found in human testicular tissue. However, conflicting data have been reported about the testicular production of DEAS ‘in vivo’. We have measured the spermatic and peripheral concentrations of testosterone (T) and DEAS in two groups of prepubertal (Group I, N = 18) and pubertal (Group II; N = 11) boys undergoing surgery for undescended testis, inguinal hernia or varicocele. Mean (± SE) spermatic concentrations of DEAS (77 ± 16 and 113 ± 19 μg/dl in Group I and II respectively) were not significantly different from peripheral concentrations (70 ± 13 and 130 ± 16 μg/dl in Group I and II respectively). Mean spermatic concentrations of T (153 ± 101 and 7515 ± 4314 ng/dl in Group I and II respectively) were significantly different from peripheral concentrations (9 ± 1 and 149 ± 53 ng/dl; P < 0.001 in both groups). Spermatic and peripheral levels of T and DEAS found in prepubertal boys were significantly lower than those found in pubertal boys. Spermatic levels of DEAS were not significantly related with spermatic levels of T in both groups. Our data show that, as in adult subjects, no significant spermatic-peripheral DEAS gradient is present in prepubertal and pubertal boys.     

An estrogen-dependent esterase activity in MCF-7 cells

The presence of steroidal esters in hormonally sensitive tissues lends importance to the esterases which convert the biologically inactive adducts to the parent potent forms. Accordingly, esterase-activities were studied in a human breast cancer model--the MCF-7 cell line. Tritiated estradiol esters- estradiol-17-acetate (EA), estradiol-17-valerate (EV) and estradiol-17-stearate (ES) were tested systematically, but 3 beta-ol esters of androgens, and phorbol diesters were also investigated. All compounds tested, except the phorbol diesters were hydrolyzed either when added to growing cultures or to the 28,000 g supernate of homogenized MCF-7 cells. Among the estrogens, the relative rates of hydrolysis were EA greater than EV greater than ES. The esterase for EA was different as it was not inhibited by saturating concentrations of EV or ES, and unlike the others its activity was stimulated by the addition of estradiol to the culture medium. The antiestrogen keoxifene,[(6-Hydroxy-2-(4-hydroxyphenyl)benzo less than b greater than thien-3-yl greater than less than 4- less than 2-(1-piperidinyl)ethoxy greater than phenyl greater than methanone], negated the stimulatory effect. Other major classes of steroids did not influence EA esterase activity. Results of inhibition experiments indicated that the esterases are of the serine active-site types. The significance of the estrogen-dependent esterase activity can be assessed when the natural substrate(s) for the enzyme is elucidated.     

Altered synthesis of heparan sulfate proteoglycans at low sulfate concentration

The structural alterations in heparan sulfate produced by sulfate deprivation were studied in cell cultures of the Engelbreth-Holm-Swarm tumor. Tumor cells were labeled in vitro with [3H]glucosamine and/or [35S]sulfate in media containing either 300 microM MgSO4 or no added carrier sulfate, and the newly synthesized proteoglycans isolated by chromatography on DEAE-Sephacel. The proteoglycans isolated from low sulfate cultures showed a reduced affinity for the column eluting at lower salt concentrations compared with the proteoglycans isolated from cultures containing sulfate, suggesting that the former were undersulfated. Analysis of the isolated heparan sulfate side chains indicated that two pools of heparan sulfate were present which differed in their degree of sulfation. Both pools were synthesized by both high sulfate and low sulfate cultures, but the highly sulfated pool was the predominant form produced in sulfate containing cultures, while the undersulfated pool was the predominant form synthesized in low sulfate cultures. The more sulfated pool contained more N-sulfate than the less sulfated pool. Few if any free amino groups were detected in either pool, suggesting that the initial deacetylation step in the biosynthesis of heparan sulfate is tightly coupled to the N-sulfation step in the cells.     

Enzymatic determination of dehydroepiandrosterone sulfate in biological fluids

A simple method is described for the determination of androgens, particularly dehydroepiandrosterone sulfate (DHEAS), in human plasma and urine. The method does not involve extraction or chromatography. An internal standard is used as reference. The androgens are enzymatically converted to estrogens and these latter compounds can be measured by the enzymatic method previously described [1]. The range of accuracy was between 99 and 103%. The precision of the method was 2.5%. The sensitivity was 0.1 pM per sample. The method is suitable for routine use, and one worker can easily perform twenty assays in one day.     

Binding of progestagens to receptor proteins in MCF-7 cells

With the aim of finding an explanation for the biological properties of progestagens currently used for contraceptive purposes, we have assessed their specificity for progesterone, androgen and oestrogen receptors in MCF-7 cells. The specificity of progestagens for the progesterone receptors in the cytosol fraction of MCF-7 cells was similar to that for progesterone receptors in human and rabbit myometrial cytosol but different from that for the progesterone receptor in rat myometrial cytosol. At 37 degrees C the relative affinity of 3-keto-desogestrel, the major metabolite of desogestrel, for the progesterone receptor in intact MCF-7 cells was twice that of levonorgestrel and Org 2058, three times that of medroxy-progesterone acetate (MPA), 4.5 times that of norethisterone and 5 times that of progesterone and cyproterone acetate whereas at 4 degrees C in the cytosol fraction of MCF-7 cells exposed to molybdate (nontransformed receptor complexes) 3-keto-desogestrel and Org 2058 displayed similar affinity. The stronger binding of 3-keto-desogestrel in intact cells was due to the higher stability of its complex with the progesterone receptor. At 37 degrees C the relative affinity of 3-keto-desogestrel for the androgen receptor in intact MCF-7 cells was half that of levonorgestrel, similar to that of norethisterone and medroxyprogesterone acetate (MPA) and at least three times higher than that of progestagens with anti-androgenic activity whereas at 4 degrees C in the cytosol fraction exposed to molybdate there was no clear difference between the relative affinities of progestagens with androgenic and anti-androgenic properties. Of the progestagens tested in this study, only norethinodrel displayed measurable but very low relative affinity for the oestrogen receptor in MCF-7 cells. We conclude that the present results of binding studies with intact MCF-7 cells correlate better with the known hormonal properties of progestagens than those obtained with the cytosol fraction exposed to molybdate at 4 degrees C.