The origin of oestrone sulphate in normal and malignant breast tissues in postmenopausal women.

Conversion of oestrone sulphate to oestrone has been suggested to make a major contribution to the level of oestrone found in breast tissues. In order to examine the ability of breast tissues to take up oestrone sulphate (E1S), 3H E1S or E1-35S was infused into postmenopausal women with advanced breast cancer. For 3 subjects infusion of 3H E1S was repeated after treatment with Danazol, a potential inhibitor of oestrone sulphatase activity. After infusion of 3H E1S significant levels of 3H E1S were detected in normal and malignant breast tissues (tissue: plasma ratios 0.14 +/- 0.13 and 0.24 +/- 0.12 respectively, mean +/- S.D., n = 5). Similar 3H E1S tissue: plasma ratios were detected after infusions of 3H E1 indicating that the 3H E1S detected in breast tissues after infusion of 3H E1S may have originated from the hydrolysis of 3H E1S in tissues other than the breast, with subsequent uptake and sulphation in breast tissues. After infusion of E1-35S no significant levels of radioactivity were detectable in normal or malignant breast tissues. Treatment with Danazol had no significant effect on tissue levels of 3H E1S or on the CRE1S E1 or MCR-E1S. It is concluded that oestrone sulphate, as such, is not taken up by breast tissues and that any contribution that oestrone sulphate makes to the oestrogen content of breast tissues will depend upon prior hydrolysis.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

Levels of androgen conjugates and oestrone sulphate in patients with breast cysts

Plasma and cyst fluid were obtained from patients with palpable breast cysts and analysed for androgen conjugates and oestrone sulphate content by radioimmunoassay. Concentrations of androgen conjugates in cyst fluids varied from 15.6 to 475.5 mumols/l. These levels were much greater than those in plasma (1.3-5.2 mumols/l) and there was no association between values in cyst aspirates and plasmas obtained from the same individuals. Levels of oestrone sulphate in breast cyst fluids (1.5-744.0 nmol/l) were also generally in excess of those in plasma (2.0-59.9 nmol/l) and again no relationship was evident between concentrations in cyst fluid and the circulation. Neither was there a relationship between levels of androgen conjugate and oestrone sulphate in plasma. In contrast, a highly significant correlation (P less than 0.001) was identified between the androgen conjugate and oestrone sulphate content of cyst fluids. Levels of both androgen conjugates and oestrone sulphate were also significantly different in groups of cysts subdivided according to electrolyte classification, cysts with low Na+:K+ ratios having higher steroid concentrations than those with high Na+:K+ ratios. The biological significance of the relationship between the two conjugates in cyst fluids remains unclear but it is suggested that the accumulation of these steroids involves a common mechanism.     

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--II. Studies with the Doluisio technique

The absorption of oestrone (E1), oestrone glucuronide (E1G) and oestrone sulphate (E1S) from the small intestine of anaesthetized rats has been evaluated using the Doluisio in situ technique. Luminal disappearance of E1 was biphasic, which is consistent with a 3-compartment model; t1/2 alpha (first phase) was less than 5 min and t1/2 beta (second phase) approx 27 min for each concentration of steroid studied (trace identical to 10 nM, 1 microM and 10 microM). In contrast, luminal disappearance of E1G and E1S was monoexponential; t1/2 for E1G was 159, 229 and 299 min (trace identical to 200 nM, 10 microM and 100 microM respectively) and for E1S, 215, 174 and 192 min (trace identical to 10 nM, 10 microM and 100 microM respectively). There was a good correlation between the luminal disappearance data and recovery of steroid in bile. Adsorption of E1S was estimated from the initial rapid fall in luminal content within the first 5 min after drug administration. The study provides further evidence that E1S can be absorbed intact. Since saccharolactone only caused a reduction in E1G absorption of 32% we also conclude that part of the administered E1G was absorbed intact.     

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--I. Importance of hydrolytic enzymes on conjugate absorption

The biliary excretion of steroid after administration of [3H]oestrone ([3H]E1), [3H]oestrone glucuronide ([3H]E1G) and [3H]oestrone sulphate ([3H]E1S) into the hepatic portal vein of anaesthetized rats was very rapid with more than 70% of E1S and greater than 80% of E1 and E1G excreted in the first 30 min. There was a lag period in the biliary excretion of E1S, this was less apparent with E1 and absent with E1G. Biliary excretion accurately reflects the amount of steroid in the portal circulation and was therefore used as an assessment of absorption from the gastrointestinal (GI) tract. Absorption (as judged by excretion in bile) was least after administration of each steroid into the stomach. The extent of absorption correlated well with the lipophilicity of the steroids as shown by their relative partition coefficients between n-octanol and pH 6.5 phosphate-buffered saline (E1 greater than or equal to E1S greater than or equal to E1G). There was no significant difference in excretion profile when the steroids were given into the caecum (at 5 h, E1, 46.3 +/- 9.1%; E1G, 42.2 +/- 14.5%; E1S, 39.9 +/- 7.1%). The similarity, despite marked differences in physicochemical properties, suggested conjugate hydrolysis to the parent steroid. In contrast, after administration into the small intestine, excretion of E1 was very rapid and was maximal at 1 h (72.5 +/- 8.0%); E1G showed a near-linear excretion rate (1 h, 14.4 +/- 3.0%; 5 h, 80.0 +/- 11.7%), whereas in comparison E1S excretion was low (1 h, 12.1 +/- 2.4%; 5 h, 36.9 +/- 2.7%). The involvement of hydrolytic enzymes in conjugate absorption was assessed. Ampicillin pretreatment (200 mg/kg/day for 2 days) reduced the absorption of E1G from both the proximal and distal small intestine (by approximately 50%) but had no effect on the absorption of E1S. There was, therefore, evidence that quantitative absorption of E1G requires prior hydrolysis (by mammalian and/or microbial enzymes) but intact absorption of E1S from this region of the tract was implicated. Ampicillin pretreatment reduced the absorption of both conjugates (greater with E1S) from the caecum; hydrolysis clearly precedes absorption from the caecum. The above findings were supported by an in vitro study which showed that ampicillin pretreatment abolished the hydrolysis of E1S by caecal contents but only partially reduced the hydrolysis of E1G. The presence of mammalian glucuronidase enzyme may account for this difference.     

Epigenetic regulation of gelsolin expression in human breast cancer cells.

Gelsolin is a multifunctional, actin-binding protein that is greatly decreased in many transformed cell lines and tumor tissues, including breast cancers. Downregulation of gelsolin RNA occurs in most breast cancers of rats, mice, and humans, but gross mutations of the gelsolin gene have not been found. Here we demonstrate by PCR and RT-PCR analysis that there are no point mutations in putative regulatory regions or the entire coding region of the cytoplasmic isoform of the gelsolin gene in human breast cancer cells (BCC). To determine if epigenetic modification is involved in downregulating gelsolin expression in MDA-MB-231 (MDA231), MCF7, and T47D BCC, we have used Southern blot analysis, 5-azacytidine (5aza) treatment, and trichostatin A (TSA) treatment. Southern blot analysis performed on genomic DNA demonstrated altered CpG methylation within intron 1 in DNA from all BCC compared to normal, mortal human mammary epithelial cells (HMEC). Treatment of the BCC with 5aza converted the DNA restriction pattern to that seen in untreated HMEC genomic DNA and caused modest increases in gelsolin RNA and protein. Incubation with TSA, an inhibitor of histone deacetylase, induced a dramatic upregulation of gelsolin RNA and protein levels which preceded apoptotic death of all BCC within 48-60 h. Our data support a role for epigenetic changes in chromatin structure leading to downregulation of gelsolin expression in human breast cancer. To our knowledge, this is the first example of a tumor suppressor gene downregulated in human breast cancer by changes in histone acetylation.     

Hormonal regulation of c-erbB-2 oncogene expression in breast cancer cells.

Expression of the c-erbB-2 (neu, HER-2) oncogene is found to be subjected to hormonal and developmental regulation in normal as well as neoplastic mammary cells. We have previously reported that estrogens inhibit c-erbB-2 expression at both the mRNA and protein level in estrogen receptor (ER)-positive, but not in ER-negative, breast cancer cell lines. Reversion of c-erbB-2 inhibition is seen with tamoxifen. The effect on c-erbB-2 expression of several other hormones and factors, which influence mammary cell growth and differentiation, has been studied. Our observations indicate that, in normal and neoplastic mammary cells, c-erbB-2 expression is inversely related to cell proliferation. While estrogens, anti-estrogens and cAMP clearly regulate c-erbB-2 mRNA levels, epidermal growth factor dramatically decreases the c-erbB-2 protein without affecting the level of c-erbB-2 mRNA. Therefore, different signals converging in terms of cell proliferation regulate c-erbB-2 expression by different molecular mechanisms.     

Estradiol-dependent regulation of angiopoietin expression in breast cancer cells

Angiopoietin-1 (Ang-1) is a ligand for Tie-2 receptors and a promoter of angiogenesis. Angiogenesis plays an important role in breast cancer, as it is one of the critical events required for tumors to grow and metastasize. In this study, we investigated the influence of estradiol (E2) on the expression of angiopoietins in breast cancer cell lines. Ang-1 mRNA and protein expressions were significantly higher in estrogen receptor-negative (ERα-) breast cancer cells than in estrogen receptor-positive (ERα+) cells. Exposure of ERα+ cells to E2 resulted in further reductions of Ang-1 levels. In mouse mammary pads inoculated with breast cancer cells, both tumor size and Ang-1 production were significantly lower in ERα+ cell-derived xenografts, as compared to those derived from ERα- cells. Reduction of circulating levels of E2 by ovariectomy eliminated this response. Overall, these results indicate that Ang-1 mRNA and protein expressions: (1) negatively correlate with the level of ERα in breast cancer cell lines; (2) are downregulated by E2 in an ERα dependent manner; and (3) positively correlate with the degree of angiogenesis in vivo. We conclude that Ang-1 is an important modulator of growth and progression of ERα- breast cancers.     

A direct assay for oestrone sulphate and its use to investigate the effect of ampicillin on plasma levels of oestrone sulphate

A direct radioimmunoassay for measuring plasma levels of oestrone sulphate has been developed using 8-anilino-2-naphthalene sulphonic acid to displace oestrone sulphate from plasma binding proteins. Oestrone sulphate was assayed by using an antiserum raised against glucuronide which cross-reacted 100% with oestrone sulphate. The direct assay gave a good analytical recovery of oestrone sulphate and there was a good correlation (r = 0.82, P less than 0.001) for plasma levels of oestrone sulphate measured by the direct assay and a method involving steroid conjugate extraction and enzyme hydrolysis. The mean (+/- S.D.) plasma level of oestrone sulphate in men was 1100 +/- 280 pg/ml. The effect of taking the antibiotic, Ampicillin, on plasma levels of oestrone sulphate was investigated in four men. Plasma levels of oestrone sulphate were significantly reduced after taking Ampicillin for 5 days. Ampicillin may act to lower plasma levels of oestrone sulphate by reducing the growth of bacteria in the gut or by inhibiting oestrogen sulphotransferase activity.     

Heregulin regulation of Akt/protein kinase B in breast cancer cells.

In the present studies, we demonstrate that heregulin is a potent and rapid activator of the serine/threonine kinase called Akt in the MCF-7 breast cancer cell line but not in 3 other breast cancer cell lines (T47D, HBL-100, and MDA-231). The extent of activation of Akt in the 4 cell lines correlated with the ability of heregulin to activate phosphatidylinositol 3-kinase and inhibition of the kinase blocked Akt activation. A monoclonal antibody to HER2 inhibited the ability of heregulin to activate Akt in the MCF-7 cells. BT474, a breast cancer cell line which overexpresses HER2, had high basal Akt enzymatic activity. This high basal activity was lowered when cells were pre-incubated with an anti-HER2 monoclonal antibody which is used to treat breast cancer patients. Our results indicate that heregulin is a potent activator of Akt and that overexpression of HER2 in breast cancers could also lead to activation of Akt.     

Estrogen regulation of cell cycle progression in breast cancer cells

Estrogens are potent mitogens in a number of target tissues including the mammary gland where they play a pivotal role in the development and progression of mammary carcinoma. The demonstration that estrogen-induced mitogenesis is associated with the recruitment of non-cycling, G₀, cells into the cell cycle and an increased rate of progression through G₁ phase, has focused attention on the estrogenic regulation of molecules with a known role in the control of G₁–S phase progression. These experiments provide compelling evidence that estrogens regulate the expression and function of c-Myc and cyclin D1 and activate cyclin E-Cdk2 complexes, all of which are rate limiting for progression from G₁ to S phase. Furthermore, these studies reveal a novel mechanism of activation of cyclin E-Cdk2 complexes whereby estrogens promote the formation of high molecular weight complexes lacking the CDK inhibitor p21. Inducible expression of either c-Myc or cyclin D1 can mimic the effects of estrogen in activating the cyclin E-Cdk2 complexes and promoting S phase entry, providing evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on the activation of cyclin E-Cdk2. These data provide new mechanistic insights into the known mitogenic effects of estrogens and identify potential downstream targets that contribute to their role in oncogenesis.     

Hormonal regulation of tumor suppressor proteins in breast cancer cells

This laboratory is studying hormonal regulation of tumor suppressor proteins, p53 and retinoblastoma (pRB). Estrogen receptor and progesterone receptor positive human breast cancer cell lines, T47D and MCF-7, were utilized for determining influence of hormonal and antihormonal agents on the level of expression of p53, state of phosphorylation of pRB, and rate of cell proliferation. The expression of p53 in T47D cells grown for 4–5 days in culture medium containing charcoal-treated (stripped) fetal bovine serum declined gradually to 10% of the level seen in control (whole serum, non charcoal-treated) groups. Supplementation of culture medium containing stripped serum with 0.1–1 nM estradiol (E₂) restored p53 to its level seen in the control within 6–24 h. Under above conditions, treatment of cells with R5020 or RU486 reduced (15–30%) the level of p53. Incubation of cells in E₂-containing growth medium caused cell proliferation and hyperphosphorylation of pRB; the latter effect was seen maximally between 24–72 h. The E₂-induced hyperphosphorylation of pRB and increase in the level of p53 were sensitive to the presence of ICI and 4-hydroxy tamoxifen (OHT). T47D and MCF-7 cells were also transiently transfected with a P1CAT reporter plasmid containing c-Myc responsive element and the levels of chloramphenicol acetyltransferase (CAT) activity were observed in response to various treatments. E₂ and OHT caused P1CAT induction as seen by increased CAT activity: E₂ caused an endogenous increase in the expression of an ICI-sensitive c-Myc form. These data suggest that estrogen upregulates p53 expression while progesterone downregulates this process. Further, E₂ regulates p53 level and pRB activity in a coordinated manner.     

Luteolytic effect of oestrone sulphate on cyclic beef heifers

The action of oestrone sulphate on luteal function was tested in cyclic beef heifers. Once daily injection of 28 or 56 mg oestrone sulphate in corn oil beginning on Day 10 of the cycle had a significant luteolytic effect as compared to corn oil-treated controls.     

Comparison of serum oestrogen concentrations in post-menopausal women taking oestrone sulphate and oestradiol.

Mean serum concentrations of oestradiol-17beta, oestrone, and oestrone sulphate in postmenopausal women were the same when measured up to six hours after treatment with either piperazine oestrone sulphate 1.5 mg or oestradiol valerate 2 mg. Maximum concentrations of oestradiol were less than those of oestrone, but oestrone sulphate reached concentrations about 30 times higher than those of oestrone. The rapid conversion of oestradiol valerate to oestrone and oestrone sulphate does not support the suggestion that in menopausal women oestradiol is less likely to be associated with a risk of endometrial carcinoma than oestrone sulphate, since the two preparations appear to become identical after ingestion.     

Modulation of oestrone sulphatase activity in breast cancer cell lines by growth factors

Currently there is much interest in the role that growth factors may play in the development of human breast tumours. We have shown previously that growth factors secreted by breast tumours may influence the activity of oestradiol hydroxysteroid dehydrogenase, the enzyme which catalyses the interconversion of oestrone (E₁) and oestradiol. As the formation of E₁ from its sulphate (E₁S) by oestrone sulphatase may be quantitatively more important than production from androstenedione via aromatase, we have studied the effect of insulin-like growth factor-1 (IGF-I) and basic fibroblast growth factor (bFGF) on oestrone sulphatase activity in the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231 breast cancer cell lines. In both these cell types, bFGF (1–200 ng/ml) and IGF-I (25–200 ng/ml) significantly stimulated oestrone sulphatase activity in a dose-dependent manner (by 8–60%) after 48 h. Additionally, cycloheximide significantly inhibited (by 90–120%) this stimulation of oestrone sulphatase activity by the two growth factors in both MCF-7 and MDA-MB-231 cells. Basal oestrone sulphatase activity was higher in the oestrogen receptor, ER - ve MDA-MB-231 cells than in the ER + ve MCF-7 breast cancer cells. We conclude that these growth factors, believed to be secreted by breast tumours, may induce enzymes of oestrogen synthesis and hence increase local production of oestrogens.     

The appearance of oestrone sulphate in the peripheral plasma of the pig early in pregnancy

Detectable concentrations of oestrone sulphate were present in 50% of the plasma samples collected from pregnant animals by Day 17. No oestrone sulphate was detected in plasma from cyclic nonpregnant pigs.     

P53 mediated regulation of metallothionein transcription in breast cancer cells

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.