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A Singh, A Purohit, M W Ghilchik, and M J Reed

Prostaglandin E2 (PGE2) and cytokines, such as interleukin-6 (IL-6) or tumour necrosis factor a (TNFalpha) can regulate aromatase activity. In the present study we have compared their abilities to stimulate aromatase activity in fibroblasts derived from 'normal' breast adipose tissue proximal to a tumour or breast tumours. PGE2, TNFalpha and IL-6 plus its soluble receptor (IL-6sR) all increased aromatase activity in these cells. Basal aromatase activity and the degree of aromatase stimulation by these factors were greater in fibroblasts derived from 'normal' breast tissue than from breast tumours. The ability of IL-6+IL-6sR to increase aromatase activity was only marginally reduced by the PG synthesis inhibitor, indomethacin, indicating that IL-6+IL-6sR does not appear to act via induction of PG synthesis. The ability of PGE2 to stimulate aromatase activity in fibroblasts derived from 'normal' breast tissue was potentiated by IL-6sR suggesting that PGE2 may act via induction of IL-6. This was confirmed by measurement of IL-6 in conditioned medium collected from these cells. A significant increase in IL-6 concentrations was detected in conditioned medium collected from cells treated with PGE2. It is concluded that in some fibroblasts PGE2 may exert part of its regulatory effect on breast tissue aromatase activity via induction of IL-6.

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L J Duncan, G V Robinson, M W Ghilchik, and M J Reed

ABSTRACT

Gross cystic breast disease frequently occurs in premenopausal women and women with apocrine cysts may have an increased risk of developing breast cancer. Many steroids, growth factors, cytokines and proteins have now been identified in breast cyst fluid (BCF), but it is not yet known whether any of these factors may be associated with an increased risk for cancer, or if these factors have a functional role within the breast. In this study we show that concentrations of the cytokine interleukin-2 are higher in BCF with a low electrolyte ratio, whereas albumin concentrations are increased in BCF with a high Na+/K+ ratio. As albumin has been shown to potentiate the ability of growth factors, such as insulin-like growth factor-I (IGF-I), to stimulate oestradiol-17β hydroxysteroid dehydrogenase (E2DH) (reductive) activity in breast cancer cells, we have examined the ability of other proteins which are found in breast secretions (α-lactalbumin, lactoferrin) or BCF (gross cystic disease proteins-15 and -24) to potentiate the action of this growth factor. While lactoferrin had no effect on E2DH, the other proteins tested significantly potentiated the effect of IGF-I on the activity of this enzyme. Recombinant human albumin also acted synergistically with IGF-I to stimulate E2DH activity, suggesting that the ability of albumin to potentiate growth factor action is an intrinsic property of the molecule itself, rather than being due to the presence of a ligand which is bound to the molecule. Results from these investigations suggest that proteins found in breast secretions and BCF may have a functional role in regulating oestrogen synthesis in breast tissues.

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Joanna M Day, Helena J Tutill, Atul Purohit, and Michael J Reed

17β-Hydroxysteroid dehydrogenases (17β-HSDs) are enzymes that are responsible for reduction or oxidation of hormones, fatty acids and bile acids in vivo, regulating the amount of the active form that is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen 17β-HSDs have been identified to date, and with one exception, 17β-HSD type 5 (17β-HSD5), an aldo–keto reductase, they are all short-chain dehydrogenases/reductases, although overall homology between the enzymes is low. Although named as 17β-HSDs, reflecting the major redox activity at the 17β-position of the steroid, the activities of these 15 enzymes vary, with several of the 17β-HSDs able to reduce and/or oxidise multiple substrates at various positions. These activities are involved in the progression of a number of diseases, including those related to steroid metabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those of aromatase and steroid sulphatase, the development of inhibitors of 17β-HSDs is at a relatively early stage, as at present none have yet reached clinical trials. However, many groups are now working on inhibitors specific for several of these enzymes for the treatment of steroid-dependent diseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in in vivo disease models. In this review, the recent advances in the validation of these enzymes as targets for the treatment of these diseases, with emphasis on 17β-HSD1, 3 and 5, the development of specific inhibitors, the models used for their evaluation, and their progress towards the clinic will be discussed.

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M J Reed, R W Cheng, P A Beranek, and V H T James

There is evidence that the free, biologically active fraction of sex steroids in the plasma, is increased in subjects with endocrine-dependent tumours. However, little is known about factors which influence the distribution of these hormones in plasma. In order to investigate this, cortisol (14 μM) was infused, or ACTH (Synacthen, 250 μg, i.v.) was injected into male subjects and this resulted in a significant decrease (up to 38% from basal values) in the fraction of testosterone not bound to sex-hormone binding globulin (non-SHBG bound testosterone). The decrease in the non-SHBG bound testosterone fraction was accompanied by an increase (up to 20% from basal values) in the non-SHBG bound oestradiol fraction after ACTH injection. No significant changes in the unbound concentrations of testosterone or oestradiol were detected. The addition of cortisol (250 nM) to a plasma pool obtained from male subjects resulted in a similar decrease in the non-SHBG bound testosterone
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M J Reed, A Purohit, L W L Woo, and B V L Potter

Introduction Steroid sulphatases regulate many important physiological processes, including the formation of neurosteroids, some aspects of reproductive function and part of the immune response. Dehydroepiandrosterone sulphatase in macrophages regulates the progression of T0 helper cells to the Th1 phenotype (Daynes et al. 1993, Rook et al. 1994) which secrete cytokines which may be involved in the development of some autoimmune diseases. However, their pivotal role in regulating oestrogen synthesis in endocrine-dependent tumours has been the greatest stimulus in developing potent steroid sulphatase inhibitors. Carlstrom et al. (1984a) made the initial observation that danazol, an isoxazole derivative of 17α-ethinyl testosterone, possessed steroid sulphatase inhibitory properties when it was found that the ratio of dehydroepiandrosterone sulphate (DHEA-S) to unconjugated DHEA increased in women treated with this drug for endometriosis. Subsequent in vitro studies confirmed the ability of danazol to inhibit DHEA sulphatase activity in breast tissues (Carlstrom et al. 1984b).
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A Purohit, L J Duncan, D Y Wang, N G Coldham, M W Ghilchik, and M J Reed

Abstract

Breast cancer remains a major cause of death in postmenopausal women. Concentrations of the biologically active oestrogen, oestradiol, are increased in breast tumours and there is now good evidence that cytokines which are present in tumours can stimulate tumour oestrogen synthesis. In this study we have examined the ability of interleukin-6 (IL-6) or tumour necrosis factor α (TNFα), either alone or in combination, to stimulate aromatase, oestradiol dehydrogenase (reductive) or oestrone sulphatase activities in cultured breast cancer cells. Both IL-6 and TNFα were able to stimulate the activities of these enzymes but in combination acted synergistically to markedly enhance enzyme activity. The possibility that α2-macroglobulin might also interact with IL-6 to enhance oestradiol dehydrogenase activity was also examined but no evidence for any synergistic interaction between these two factors was obtained. Cytokines, such as IL-6 and TNFα, are emerging as having a central role in regulating breast tumour oestrogen synthesis. Understanding the role that cytokines have in regulating oestrogen synthesis in breast tumours should lead to the development of novel therapeutic agents for use in the treatment of women with breast cancer.

Endocrine-Related Cancer (1997) 4 323-330

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M J Reed, A Angeli, J H Thijssen, A Milewicz, I Számel, J Huber, A Harlozinska-Szmyrka, J Tóth, and J Kornafel

INTRODUCTION During the last 15-20 years several research groups, in Europe and North America, have been analysing the biochemical composition of breast cyst fluid (BCF). Cysts occur frequently in the premenopausal breast and gross cystic breast disease (GCBD), defined as the presence of cysts more than 1 cm in size and detectable by clinical examination, is a common condition, affecting 7-10% of women in the western world (Haagensen et al. 1981). There are several reasons for analysing the composition of BCF, which can be readily aspirated for biochemical analysis. Although the topic is controversial, the presence of GCBD is said to be associated with an increased risk of developing breast cancer, and by the analysis of BCF it may be possible to identify a marker of the subsequent risk. If GCBD is associated with an increased risk of breast cancer, the characterisation of steroids, growth factors, cytokines and
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Joanna M Day, Paul A Foster, Helena J Tutill, Fabien Schmidlin, Christopher M Sharland, Jonathan D Hargrave, Nigel Vicker, Barry V L Potter, Michael J Reed, and Atul Purohit

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyse the 17-position reduction/oxidation of steroids. 17β-HSD type 3 (17β-HSD3) catalyses the reduction of the weakly androgenic androstenedione (adione) to testosterone, suggesting that specific inhibitors of 17β-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia. STX2171 is a novel selective non-steroidal 17β-HSD3 inhibitor with an IC50 of ∼200 nM in a whole-cell assay. It inhibits adione-stimulated proliferation of 17β-HSD3-expressing androgen receptor-positive LNCaP(HSD3) prostate cancer cells in vitro. An androgen-stimulated LNCaP(HSD3) xenograft proof-of-concept model was developed to study the efficacies of STX2171 and a more established 17β-HSD3 inhibitor, STX1383 (SCH-451659, Schering-Plough), in vivo. Castrated male MF-1 mice were inoculated s.c. with 1×107 cells 24 h after an initial daily dose of testosterone propionate (TP) or vehicle. After 4 weeks, tumours had not developed in vehicle-dosed mice, but were present in 50% of those mice given TP. One week after switching the stimulus to adione, mice were dosed additionally with the vehicle or inhibitor for a further 4 weeks. Both TP and adione efficiently stimulated tumour growth and increased plasma testosterone levels; however, in the presence of either 17β-HSD3 inhibitor, adione-dependent tumour growth was significantly inhibited and plasma testosterone levels reduced. Mouse body weights were unaffected. Both inhibitors also significantly lowered plasma testosterone levels in intact mice. In conclusion, STX2171 and STX1383 significantly lower plasma testosterone levels and inhibit androgen-dependent tumour growth in vivo, indicating that 17β-HSD3 inhibitors may have application in the treatment of hormone-dependent prostate cancer.

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S Krajewski, M Krajewska, B C Turner, C Pratt, B Howard, J M Zapata, V Frenkel, S Robertson, Y Ionov, H Yamamoto, M Perucho, S Takayama, and J C Reed

Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer.