Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines

Breast cancers require the presence of estrogens for the maintenance of growth at some time in the course of their development, as does normal breast tissue. Sulfation is an important process in the metabolism and inactivation of steroids, including estrogens, because the addition of the charged sulfonate group prevents the binding of the steroid to its receptor. Also, many of the therapeutic and chemopreventive agents used in the treatment of breast cancer are substrates for the sulfotransferases (STs). The activity and expression of four cytosolic STs, which are the human phenol-sulfating and monoamine-sulfating forms of phenol ST (PST), dehydroepiandrosterone ST, and estrogen ST (hEST), were assayed in normal breast cells and in breast cancer cell lines. ST activities and immunoreactivities were assayed in the estrogen receptor-positive human breast cancer cell lines ZR-75-1, T-47D and MCF-7; in the estrogen receptor-negative breast cancer cell lines BT-20, MDA-MB-468, and MDA-MB-231; and in normal human mammary epithelial cells. The PSTs were the most highly expressed ST activities in the breast cancer cell lines, although the levels of activity varied significantly. ZR-75-1 and BT-20 cells possessed the highest levels of activity of the human phenol-sulfating form of PST. The breast cancer cell lines showed only trace levels of dehydroepiandrosterone ST and hEST activities. In contrast, hEST was the only ST detectable in human mammary epithelial cells. Understanding the regulation of ST activity in these breast cancer and normal breast cell lines will improve our knowledge of the role of sulfation in breast cancer and provide a model with which to study the mechanism of action of estrogens in mammary cells.     

Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer

The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.     

Role of 17 beta-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17 beta-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17 beta-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17 beta-HSD isozymes have been characterized so far, and the data strongly suggests that different 17 beta-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17 beta-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17 beta-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17 beta-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17 beta-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17 beta-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17 beta-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

Androgens down-regulate bcl-2 protooncogene expression in ZR-75-1 human breast cancer cells

Although a large proportion of primary human breast cancers express the androgen receptor, and treatment with androgens exerts beneficial effects in women with breast cancer, the role and especially the mechanism of action of androgens in breast cancer development and growth are not well understood. The potential effect of androgens on bcl-2 protooncogene expression was investigated in a human breast cancer cell line whose proliferation is known to be inhibited by androgens. The estrogen-responsive ZR-75-1 cells were grown in the presence or absence of 5alpha-dihydrotestosterone (DHT), alone or in combination with 17beta-estradiol. DHT caused a marked down-regulation of Bcl-2 protein and messenger RNA levels in both the presence and absence of 17beta-estradiol. The inhibitory effect of DHT was completely prevented by coincubation with the pure antiandrogen hydroxyflutamide. The present data indicate that androgens can down-regulate bcl-2 protooncogene levels via an androgen receptor-mediated mechanism, thus providing a novel mechanism for their known inhibitory effect on breast cancer cell growth.     

The effects of IL-6 on cell adhesion and e-cadherin expression in breast cancer

Interleukin 6 (IL-6) is a pleiotropic inflammatory cytokine and its role in cancer is not yet clear. The effects of IL-6 on four breast cancer cell lines and normal mammary epithelium, cultured from milk were tested. Four different patterns of response to IL-6 were found depending on the differentiation status of the cells. In normal mammary epithelial cultures, the effects of IL-6 were mainly growth inhibitory, whereas in MCF-7, IL-6 had growth inhibitory and anti-adhesive effects. In T-47D and ZR-75-1 the anti-adhesive effects were prominent although the growth inhibitory effects were not. These anti-adhesive effects were associated with epithelioid to fibroblastoid morphological changes and a local decrease in E-cadherin expression. In the highly invasive cell line MDA-MB-231, which does not express E-cadherin, no effects of IL-6 were seen. IL-6 levels in the serum of 60 breast cancer patients were found to be increased in 27% (16/60) compared to 2% (1/50) in a control group. Furthermore, it was found that altered E-cadherin expression was seen in 69% of the primary tumours, although no significant association was found between raised serum IL-6 levels and altered E-cadherin expression. Finally IL-6 serum levels did not effect the survival of breast cancer patients. The authors therefore implicate IL-6 as a possible factor important in breast cancer progression and metastasis formation, although the clinical significance of this cytokine in breast cancer patients could not be established.     

Interleukin 6 acts as a paracrine growth factor in human mammary carcinoma cell lines

The effect of interleukin 6 (IL-6) on normal and human mammary carcinoma epithelial cells was studied. IL-6 inhibited the growth of estrogen receptor-positive [ER(+)] breast cancer cell lines, which underwent apoptosis with prolonged treatment. In contrast, ER(-) breast cancer cell lines were resistant to IL-6-mediated growth inhibition. By examining the components of the IL-6 receptor (IL-6R) system, we found that ER(+) breast cancer cells expressed predominantly soluble IL-6Ralpha, whereas the ER(-) breast cancer cells expressed primarily the transmembrane form of the IL-6R, gp130. In addition, detectable levels of IL-6 were secreted into the medium by ER(-) but not ER(+) breast cancer cells. Furthermore, the supernatant obtained from IL-6-secreting, ER(-) cells suppressed the growth of IL-6-sensitive, ER(+) breast cancer cells in a paracrine fashion. Although IL-6 is secreted by ER(-) breast cancer cells, this cytokine does not seem to stimulate the proliferation of these cells in an autocrine fashion. These studies indicate that IL-6 can regulate the growth of normal and transformed human mammary epithelial cells differentially, and that IL-6 secretion by some ER(-) breast cancer cells can function as a paracrine growth factor, suppressing the growth of ER(+) breast cancer cells in vitro.     

Arimidex: a potent and selective fourth-generation aromatase inhibitor

Dehydroepiandrosterone-sulfate (DHEA-S), the main secretory product of the human adrenal, requires the presence of steroid sulfatase, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), 5 alpha-reductase, and aromatase to form the active androgen dihydrotestosterone (DHT) and the estrogens 17 beta-estradiol (E2) and 5-androst-ene-3 beta,17 beta-diol (delta 5-diol) in peripheral target tissues. Because humans, along with non-human primates are unique in having adrenals that secrete large amounts of DHEA-S, the present study investigated the tissue distribution of the enzymatic activity of the above-mentioned steroidogenic enzymes required for the formation of active sex steroids in the male and female rhesus monkey. Estrone and DHEA sulfatase activities were measured in all 25 tissues examined, and with the exception of the salivary glands, estrogenic and androgenic 17 beta-HSDs were present in all the tissues examined. The adrenal, small and large intestine, kidney, liver, lung, fat, testis, prostate, seminal vesicle, ovary, myometrium, and endometrium all possess the above-mentioned enzymatic activities, thus suggesting that these tissues could possibly form the biologically active steroids E2 and DHT from the adrenal precursor DHEA-S. On the other hand, the oviduct, cervix, mammary gland, heart, and skeletal muscle possess all the enzymatic activities required to synthesize E2 from DHEA-S. The present study describes the widespread tissue distribution of steroid sulfatase, 3 beta-HSD, 17 beta-HSD, 5 alpha-reductase, and aromatase activities in rhesus monkey peripheral tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human bone cells stimulate the growth of human breast carcinoma cells

Human breast cancer cells were cultured together with their metastatic target, bone tissue, to analyze possible growth promotion effects. The coculture of human osteosarcoma cells (TE-85) with human mammary carcinoma cells (ZR-75.1) resulted in up to 8.4-fold stimulation of proliferation of the breast tumor cells. Cell contact of the two cultures was permitted through the channels of Nuclepore filters. However, physical contact turned out not to be necessary, since the proliferative stimulus was also mediated by a bone-derived diffusible factor. Conditioned medium (CM), collected from human primary bone cultures, enhanced the rate of proliferation of several breast tissue cell lines (ZR-75.1, BT-20, HBL-100), while some lines were not affected by osteoblast CM. Breast tissue lines responding to bone CM express low to intermediate levels of the c-erbB-2 gene, in contrast to nonstimulated lines, which overexpress the gene. Recent observations of metastatic spread in breast cancer patients suggest a distinctive pattern of secondary tumor distribution in association with c-erbB-2 protein expression. Bone tissue seems to be a preferential target for metastases of c-erbB-2-negative breast tumors.     

Steroid hormone regulation of cytokine secretion by proteolipid protein-specific CD4+ T cell clones isolated from multiple sclerosis patients and normal control subjects

Steroid hormones have long been known to modulate immune function, and recent studies indicate that one of the means by which they do so involves effects on the secretion of immunoregulatory cytokines. Our laboratory has found recently that estradiol (E2) selectively modifies cytokine secretion in proteolipid protein (PLP)-specific, CD4+ T cell clones isolated from patients with the demyelinating disease, multiple sclerosis, and from normal control subjects. The data suggest that E2 may play a role in regulating the balance between pro- and antiinflammatory conditions, especially at concentrations typical of pregnancy. To determine whether other pregnancy-associated steroid hormones are capable of similar activity, we expanded our testing to include estrone (E1), estriol (E3), progesterone, and dexamethasone. The results indicate that E1 and E3 enhance secretion of Ag- or anti-CD3-stimulated IL-10 and IFN-gamma in dose-dependent fashion, almost identical to that of E2. The effect on IL-10 was more potent than occurred with IFN-gamma. In addition, E1 and E3, like E2, had a biphasic effect on TNF-alphabeta secretion, with low concentrations stimulatory, and high doses inhibitory. None of the estrogens influenced IL-4 or TGF-beta secretion. Progesterone enhanced secretion of IL-4, without affecting any other tested cytokine. Finally, dexamethasone induced TGF-beta secretion, but inhibited IFN-gamma and TNF-alphabeta. This differential effect of steroid hormones on the secretion of cytokines by CD4+ human T cell clones is consistent with the possibility that, collectively, they promote antiinflammatory conditions at high concentrations typical of pregnancy.     

Intratumoral aromatase in human breast, endometrial, and ovarian malignancies

In human estrogen-dependent neoplasms such as breast, endometrioid endometrial, and surface epithelial-stromal ovarian carcinomas, intratumoral aromatase is considered to play important roles in converting circulating androgens derived from adrenal cortex and/or ovary to estrogens, possibly in association with 17 beta-HSD type 1 and estrogen sulfatase. Analysis of intratumoral aromatase in these estrogen-dependent neoplasms is important not only in understanding the development and biological behavior of these tumors, but also in the clinical management of these patients, because suppression of intratumoral aromatase by newly developed aromatase inhibitors may provide new potentials in endocrine therapy of these patients.     

Profound suppression of plasma estrogens by megestrol acetate in postmenopausal breast cancer patients

Twelve postmenopausal women suffering from advanced breast cancer had plasma estrogens, androgens, cortisol, and gonadotropins determined before therapy and during treatment with megestrol acetate (MA) in oral doses escalated from 40 to 160 mg. The plasma clearance and production rate of estrone and estrone sulfate were determined before treatment and after 4 weeks of therapy with 160 mg MA. Treatment with MA suppressed plasma levels of dehydroepiandrosterone sulfate, androstenedione, and cortisol in a dose-dependent manner to <10% of pretreatment values. Plasma testosterone, estradiol, estrone, and estrone sulfate were suppressed to 18-29% of pretreatment values, whereas the gonadotropins were suppressed to 35-52%. The plasma clearance rates of estrone and estrone sulfate were increased by a mean value of 23.7% (P < 0.01) and 23.5% (P < 0.025), whereas the production rates were reduced by 76.7% (P < 0.0005) and 76.1% (P < 0.0005), respectively. Our findings indicate that MA causes profound suppression of adrenal steroid production but in addition suppresses ovarian secretion of androgens in postmenopausal breast cancer patients. The reduction in plasma estrogens is comparable to values obtained with commonly used aromatase inhibitors and may be responsible for its antitumor effects in breast cancer.