Antiestrogenic activities of Ginkgo biloba extracts

Most climacteric and postmenopausal women appear to have vasomotor symptoms as well as a high risk of osteoporosis and cardiovascular disease. Although exogenous estrogens can reduce these symptoms, women are reluctant to use hormone replacement therapy (HRT) due to its undesirable side effects, such as irregular bleeding and an increased risk of breast cancer. A previous study suggested that Ginkgo biloba extracts (GBE) have estrogenic activity and might be suitable as an alternative to HRT. However, there are no reports of the preventive effect of GBE on breast cancer, which is the side effect of classical HRT. In this study, it was confirmed that GBE exhibits estrogenic and antiestrogenic activity depending on the E₂ and GBE concentration, via estrogen receptor (ER)-dependent and ER-independent pathways. In addition, GBE reduced the E₂ levels by stimulating the E₂ metabolism and inhibiting E₂ synthesis, which indicates that GBE can induce antiestrogenic activity via the depletion of E₂. Furthermore, GBE might have similar action to selective arylhydrocarbon receptor modulators (SAhRMs), which induce antiestrogenic activity through cross-talk between the arylhydrocarbon receptor (AhR) and ER. In conclusion, GBE has a biphasic effect on estrogen, and can be considered as a potential alternative to HRT with chemopreventive effects on breast cancer. However, further studies on animals and humans will be required.     

The role of estrogen in the initiation of breast cancer

Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17β-estradiol (E₂) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E₂, 4-OH-E₂, and 16--OH E₂), respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF-10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP). Neither Tamoxyfen (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E₂ induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER- and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E₂ induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E₂, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.     

Estrogens,apoptosis and cells of neural origin

In mammals, estrogens have a multiplicity of effects in all known neural cells. We review here some of the mechanisms enabling estrogens to differentiate their influence on neural targets. In view of the potential interest in the use of estrogens in the therapy of several pathologies of the nervous system, we have proposed the use of a reductionist approach for the systematic understanding of estrogen activities in each specific type of target cell. We have therefore generated a model system in which to study the activation of one of the known estrogen receptors: estrogen receptor alpha. This system allowed us to identify a number of novel genes, the expression of which may be influenced following the activation of this receptor subtype by estradiol. We here report on preliminary data obtained by the study of one of these target genes, nip2, which encodes a proapoptotic protein product. We hypothesize that Nip2 might be an important molecular determinant for estrogen anti-apoptotic activity in cells of neural origin.     

Estrogen metabolism and formation of estrogen-DNA adducts in estradiol-treated MCF-10F cells. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction and catechol-O-methyltransferase inhibition

Formation of estrogen metabolites that react with DNA is thought to be a mechanism of cancer initiation by estrogens. The estrogens estrone (E₁) and estradiol (E₂) can form catechol estrogen (CE) metabolites, catechol estrogen quinones [E₁(E₂)-3,4-Q], which react with DNA to form predominantly depurinating adducts. This may lead to mutations that initiate cancer. Catechol-O-methyltransferase (COMT) catalyzes an inactivation (protective) pathway for CE. This study investigated the effect of inhibiting COMT activity on the levels of depurinating 4-OHE₁(E₂)-1-N3Ade and 4-OHE₁(E₂)-1-N7Gua adducts in human breast epithelial cells. MCF-10F cells were treated with TCDD, a cytochrome P450 inducer, then with E₂ and Ro41-0960, a COMT inhibitor. Estrogen metabolites and depurinating DNA adducts in culture medium were analyzed by HPLC with electrochemical detection. Pre-treatment of cells with TCDD increased E₂ metabolism to 4-OHE₁(E₂) and 4-OCH₃E₁(E₂). Inclusion of Ro41-0960 and E₂ in the medium blocked formation of methoxy CE, and depurinating adducts were observed. With Ro41-0960, more adducts were detected in MCF-10F cells exposed to 1 μM E₂, whereas without the inhibitor, no increases in adducts were detected with E₂ ≤ 10 μM. We conclude that low COMT activity and increased formation of depurinating adducts can be critical factors leading to initiation of breast cancer.     

Interactions between FSH, estradiol-17β and transforming growth factor-β regulate growth and differentiation in the rat gonad

Estradiol-17β (E₂) is a mitogen in vivo for the proliferation of granulosa cells in the rat ovary. E₂ is synthesized by the preovulatory follicle through a series of gonadotrophin-dependent events: LH stimulates thecal cells to synthesize androgens (androstenedione and testosterone) which are substrates for FSH-induced aromatization to estrogens in granulosa cells. More recently, we have found that transforming growth factor-β (TGF-β) stimulates DNA synthesis in rat granulosa cells in vitro and this effect is augmented by FSH. Since E₂ is a mitogen in vivo and TGF-β is the only known growth factor to stimulate proliferation in vitro, the possible link between the actions of E₂ and TGF-β were examined. E₂ stimulated the secretion of a TGF-β-like factor by rat granulosa cells in culture, and with time DNA synthesis was stimulated. The mitogenic action of E₂ was enhanced in the presence of FSH, and attenuated by a neutralizing antibody to TGF-β. The latter observations have identified TGF-β as the “missing-link” in the mitogenic actions of E₂ on rat granulosa cells. In addition to the growth-promoting actions of TGF-β plus FSH, TGF-β enhanced FSH-induced aromatase activity. Consequently, FSH plus TGF-β stimulates both the proliferation and aromatization capacity of rat granulosa cells. We propose that interactions between FSH, E₂ and TGF-β lead to the exponential increase in serum E₂ levels that occurs during the follicular phase of the cycle. Similarly, FSH stimulates the aromatization of exogenous androgens to estrogen by Sertoli cells isolated from immature rat testes, and there is a correlation between FSH-induced aromatization and mitotic activity. We have shown that FSH plus TGF-β stimulates DNA synthesis in Sertoli cells. Since E₂ increases the secretion of TGF-β by Sertoli cells, interactions between FSH, E₂ and TGF-β may provide the mitogenic stimulus for Sertoli cells during the prepubertal period. In summary, our findings suggest that the estrogen-induced growth of rat granulosa cells is mediated through the production of TGF-β, which acts as an autocrine regulator of proliferation. We also propose that the growth-promoting actions of FSH on Sertoli cells may depend upon a cascade series of events involving estrogens and TGF-β.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Estrogen stimulation of creatine kinase B specific activity in 3T3L1 adipocytes after their differentiation in culture: Dependence on estrogen receptor

We have demonstrated previously that rat adipose tissue showed sex and depot-specific responses to gonadal steroids. The epididymal fat pad in males responded exclusively to androgens by increased specific activity of the brain type isozyme of creatine kinase (CK). In females, the parametrial adipose tissue responded exclusively to estrogens. The present study was undertaken to follow the responsiveness to steroid hormones, and the presence of estrogen receptors (ER), in 3T3L1 cells during their differentiation from pre-adipocytes to adipocytes. In pre-adipocytes in which the basal CK specific activity is low, there was no CK response to 17β estradiol (E₂) or dihydrotestosterone (DHT). Differentiation of the cells into adipocytes was accompanied by increased basal CK activity which was stimulated by E₂, but not by DHT. Responsiveness to E₂ began 5 days after switching pre-adipocytes to differentiation medium. Upon differentiation, ER became demonstrable in the cell nuclei by staining with FITC labeled anti-idiotypic antibody (clone 1D5) directed against the steroid binding domain of ER. The response to E₂ was time-dependent and blocked completely by cycloheximide or actinomycin D. 1D5 itself, which has an estrogen mimetic effect, stimulated CK activity in the cells similarly to E₂. The antiestrogen tamoxifen which also stimulated CK activity in the adipocytes, completely blocked E₂ action. The ‘pure’ antagonist of E₂, ICI 164,384 and the tissue-selective antiestrogens, raloxifene or tamoxifen methiodide were also complete antagonists with no agonistic effects. The response of the 3T3L1 adipocytes to E₂ was upregulated by 1,25(OH)₂D₃. Moreover, IGF1 was also a potent stimulator of CK in these cells, and therefore may mediate partially the stimulation by E₂. Transient transfection of the pre-adipocytes with ER permitted E₂ induction of CK. Thus, the appearance of ER and concomitant responsiveness to E₂ is another hormone-related change occurring in 3T3L1 cells during differentiation, in addition to changes such as development of insulin responsiveness. The interactions in this system provide a useful in vitro model for investigating the development of responsiveness to E₂.     

DT56a (Femarelle): a natural selective estrogen receptor modulator (SERM)

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17β (E₂), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E₂ but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E₂, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E₂ stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E₂ stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E₂'s stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E₂ stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E₂ was completely abolished by DT56a and E₂-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E₂, but when given simultaneously, at supra physiological doses, inhibited these E₂'s effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.     

An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E₁), estradiol (E₂) and estrone sulfate (E₁S) levels in the ultra-low range. Following incubation with [³H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E₁S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E₁, E₂) were separated by chromatography (LH-20). Estrone and E₁S (following hydrolysis) were converted into E₂, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[¹²⁵I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E₁, E₂ and E₁S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E₁, 0.67 pmol/L for E₂, and 0.55 pmol/L for E₁S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E₁, 5.1% for E₂ and 6.1% for E₁S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E₁, E₂, and E₁S, respectively. Considering normal plasma levels for E₂ (15 pmol/L), E₁ (80 pmol/L) and E₁S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E₂, E₁ and E₁S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Emerging principles for the development of resistance to antihormonal therapy: implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E₂ and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E₂), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E₂. Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E₂ in Phase II antihormone-resistant tumors reversed the E₂-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E₂-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E₂ and fulvestrant.     

Serum steroid hormones, sex hormone-binding globulin concentrations, and urinary hydroxylated estrogen metabolites in post-menopausal women in relation to daidzein-metabolizing phenotypes

Equol and O-desmethylangolensin (O-DMA) are products of bacterial metabolism of daidzein, an isoflavone in soybeans; thus, the presence or absence of equol and/or O-DMA in urine is a marker of particular intestinal bacteria profiles. Plasma hormone concentrations may be lower in pre-menopausal women who harbor the bacteria capable of producing equol (equol producers) compared to women who do not (equol non-producers). We evaluated concentrations of serum hormones, sex hormone-binding globulin (SHBG), and urinary 2-hydroxyestrone (2-OH E₁) and 16-hydroxyestrone (16-OH E₁) in relation to equol-producer and O-DMA-producer phenotypes in 89 post-menopausal women. Follicle stimulating hormone (FSH) was 23% greater in O-DMA-producers compared to non-producers (P=0.04). No significant differences in serum estrogens, androgens, metabolic hormones, or SHBG were observed in relation to either daidzein-metabolizing phenotype. Compared with non-producers within each phenotype, age-adjusted 2-OH E₁:16-OH E₁ was 27% greater (P=0.06) in equol-producers and 9% greater (P>0.10) in O-DMA-producers, and 2-OH E₁ concentrations were 24% greater in equol producers (P=0.07) and 42% greater in O-DMA producers (P=0.02). No significant differences in 16-OH E₁ were observed in relation to either phenotype. These results suggest that interindividual variability in intestinal bacteria may be related to differences in products of hormone metabolism in post-menopausal women.     

17β-Hydroxysteroid dehydrogenase activity in endometrial cancer cells: Different metabolic pathways of estradiol in hormone-responsive and non-responsive intact cells

In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E₂) as precursor resulted in: (1) elevated formation (up to 90%) of E₂-sulphate (E₂-S), using lower precursor concentrations; (2) very limited conversion to estrone (E₁) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E₂-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E₁ production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E₁ (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E₂ metabolism could be minor in Ishikawa cells as a consequence of the high rate of E₂-S formation encountered is contradicted by the evidence that conversion to E₁ also remains limited in the presence of much lower E₂-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17β-hydroxysteroid dehydrogenase (17β-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E₂) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17β-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types.     

Effects of anastrozole on the intratumoral gene expression in locally advanced breast cancer

Intratumoral levels of E₁ (oestrone), E₁S (oestrone sulphate) and E₂ (oestradiol) are significantly reduced by treatment with the aromatase inhibitor anastrozole regardless of treatment response. The purpose of the present pilot study was to look for additional markers of biochemical response to aromatase inhibitors on mRNA expression level. Whole genome expression was studied using microarray analysis of breast cancer tissue from 12 patients with locally advanced tumors, both before and following 15 weeks of treatment with the aromatase inhibitor anastrozole (Arimidex^®). Intratumoral mRNA levels for a subset of genes coding for steroid metabolizing enzymes, hormone receptors and some growth mediators involved in cell cycle control were analysed by quantitative RT-PCR. There was a correlation between the two methods for some but not all genes. The mRNA expression levels of the different genes were correlated to each other and to the intratumoral levels of E₁, E₂ and E₁S, before and after the treatment. Notably, a correlation of the E₁/E₂ metabolic ratio to the mRNA levels of CYP19A1 was observed before treatment (r = 0.745, p < 0.005). Whole genome expression analysis of these 12 breast cancer patients revealed similar tumor classification to previously published larger studies. Tumors with no or low expression of ESR1 (oestrogen receptor) clustered together and were characterized by a strong basal-like signature highly expressing keratins 5/17, cadherin 3, frizzled and apolipoprotein D, among others. The luminal epithelial tumor cluster, on the other hand, highly expressed ESR1, GATA binding protein 3 and N-acetyl transferase. An evident ERBB2 cluster was observed due to the marked over-expression of the ERBB2 gene and GRB7 and PPARBP in this patient material). Using significance analysis of microarrays (SAM), we identified 298 genes significantly differently expressed between the partial response and progressive disease groups.     

Plasma estrogen suppression with aromatase inhibitors evaluated by a novel, sensitive assay for estrone sulphate

Aromatase inhibition is a well-defined treatment option for postmenopausal breast cancer. Although several aromatase inhibitors such as aminoglutethimide, formestane, fadrozole have been found to inhibit in vivo aromatization by>85%, previous studies reported plasma estrogen levels to be sustained at approximately 20–50% of their control level during treatment with these drugs. The discrepancy could be due to lack of sensitivity or non-specific crossreactions in the radioimmunoassay (RIA) methods. Mean plasma levels of estrone (E₁) and estradiol (E₂) in postmenopausal women are approximately 80 and 20 pmol/l, respectively; on the contrary, mean plasma levels of the estrogen conjugate estrone sulphate (E₁S) are approximately 4–500 pmol/l. Most RIA methods for plasma E₂ and E₁ measurements have sensitivity limits in the range of 2–3 and 7–10 pmol/l, respectively; accordingly, the suppression of plasma estrogens by more than 80–90% will produce hormone values below the sensitivity limit of the method in many patients. Recently, we developed a new method to determine plasma E₁S. This assay has a sensitivity limit of 2.7 pmol/l. In theory, this method may allow the determination of plasma E₁S levels suppressed to less than 2% of control values in the majority of patients. Using this method, we found different aromatase inhibitors such as formestane, aminoglutethimide, formestane and aminoglutethimide administered in concert or anastrozole to suppress plasma E₁S levels down to 24, 13, 7 and 4%, respectively. The suppression of plasma E₁S evaluated with this method thus approaches the percentage aromatase inhibition measured with tracer studies.