Synergistic interaction of growth factors and albumin in regulating estradiol synthesis in breast cancer cells
Abstract
Estradiol 17β-hydroxysteroid dehydrogenase acts to convert estrone to the biologically active estrogen, estradiol, in breast tumors and MCF-7 breast cancer cells in vitro. In this study we have examined the ability of albumin to influence the effect of growth factors (insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), transforming growth factor-α (TGFα) and cytokines (interleukin (IL)-1, IL-6) on estradiol 17β-hydroxysteroid dehydrogenase activity in MCF-7 cells. IGF-I (80 ng/ml) or albumin (30 μg/ml) stimulated estradiol 17β-hydroxysteroid dehydrogenase activity by 144% and 102% (p < 0.01). The combination of IGF-I and albumin, however, produced a marked (704%) synergistic stimulation of estradiol 17β-hydroxysteroid dehydrogenase activity. EGF or TGFα failed to stimulate estradiol 17β-hydroxysteroid dehydrogenase activity and no synergism with albumin was detected. IL-1 (10 ng/ml), but not IL-6, also stimulated estradiol 17β-hydroxysteroid dehydrogenase activity and acted synergistically with albumin to stimulate enzyme activity. MCF-7 cells were shown to specifically bind 125I-albumin and binding is increased by pretreatment of cells with IGF-I (80 ng/ml) for 48 h.
It is concluded that the synergism that results from treating MCF-7 cells with albumin and IGF-I may result from increased albumin uptake and subsequent biological effect.
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The regulation and inhibition of 17β-hydroxysteroid dehydrogenase in breast cancer
2006, Molecular and Cellular Endocrinology17β-Hydroxysteroid dehydrogenase Type 1 (17β-HSD1) has a pivotal role in regulating the synthesis of oestradiol (E2) within breast tumours. In whole body studies in postmenopausal women with breast cancer the conversion of oestrone (E1) to E2 (4.4 ± 1.1%) was much lower than the inactivation of E2 to E1 (17.3 ± 5.0%). In contrast, an examination of in vivo oestrogen metabolism within breast tumours revealed that whereas little metabolism of E2 occurred, E1 was converted to E2 to a much greater extent in malignant (48 ± 14%) than in normal (19 ± 6%) breast tissue. Findings from these studies originally suggested that oestrogen metabolism within breast tumours may differ from the mainly oxidative direction found in most other body tissues and that the activity of 17β-HSD1 might be regulated by tumour-derived factors. Several growth factors (e.g. IGF-I, IGF-II) and cytokines (e.g. IL-6, TNFα) have now been identified which can markedly stimulate the activity of 17β-HSD1 and such a mechanism may account for the high concentrations of E2 found in most breast tumours. Cells of the immune system, which can infiltrate breast tumours, are thought to be a major source of the growth factors and cytokines which can modulate 17β-HSD1 activity.
Given the central role that 17β-HSD1 has in regulating breast tumour E2 concentrations the development of potent inhibitors of this enzyme has recently attracted considerable attention. Our initial studies in this area explored the use of derivatives of E1 as inhibitors, with 2-ethyl- and 2-methoxy E1 being found to inhibit 17β-HSD1 activity in T-47D breast cancer cells by 96 ± 2 and 91 ± 1% respectively at 10 μM, but with a lack of specificity. Using the E1 scaffold a number of potent, selective 17β-HSD1 inhibitors have now been identified including E1- and 2-ethyl-E1 containing a side chain with a m-pyridylmethylamidomethyl functionality extending from the 16β position of the steroid nucleus. At 10 μM these compounds both inhibited 17β-HSD1 activity by >90%, however some inhibition of 17β-HSD2 activity was exhibited by the E1 derivative (25%) but not the 2-ethyl analogue. It is now apparent that 17β-HSD1 activity contributes to the high E2 concentrations found in most breast tumours. The identification of potent, selective novel 17β-HSD1 inhibitors will allow their efficacy to be tested in in vitro and in vivo studies.
Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERα in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERα specific monoclonal antibody also revealed specific, multiple Mr mERs (67, 110, and 130 k Mr). In addition, Western blot analysis with an ABP antiserum revealed a potential 60 k Mr ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60 k Mr ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.
The role and proposed mechanism by which oestradiol 17β-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations
1995, Journal of Steroid Biochemistry and Molecular BiologySynthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17β-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor α (TNFα), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNFα, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.
The role of cytokines and sulphatase inhibitors in regulating oestrogen synthesis in breast tumours
1995, Journal of Steroid Biochemistry and Molecular BiologySynthesis of oestrogens within breast tissues makes an important contribution to the high concentrations of oestradiol which are found in breast tumours. The activities of the enzymes involved in oestrogen synthesis, i.e. the aromatase, oestradiol dehydrogenase (E2DH) and oestrone sulphatase (E1-STS), can be stimulated by several growth factors and cytokines. As it is possible that some of these factors may be derived from cells of the immune system (macrophages and lymphocytes), the effects of basic fibroblast growth factor (bFGF) and interleukin-2 (IL-2), which are produced by these cells, on E2DH activity was examined in MCF-7 cells. Treatment of these cells with bFGF resulted in a dose-dependent increase in E2DH reductive activity whereas IL-2 was inactive at the concentration tested. To obtain further evidence that factors produced by macrophages and lymphocytes can modulate the activities of enzymes involved in oestrogen synthesis, conditioned medium was collected from these cells and found to stimulate both E1-STS and E2DH activities. In addition to understanding the control of oestrogen synthesis in breast tumours an inhibitor to block the synthesis of oestrone via the oestrone sulphatase pathway was developed. Oestrone-3-O-sulphamate (EMATE) is a potent, irreversible, inhibitor of E1-STS. A single dose of EMATE (10 mg/kg) inhibited tissue E1-STS activity in rats by more than 95% for up to 7 days, indicating that this compound may have considerable therapeutic potential for the treatment of breast cancer. Evidence is also reviewed that another steroid sulphatase, dehydroepiandrosterone sulphate sulphatase, may have a crucial role in regulating cytokine production and that this may indirectly control tumour oestrogen synthesis.
Stimulation of aromatase activity in breast fibroblasts by tumor necrosis factor
1994, Molecular and Cellular EndocrinologyThe conversion of androstenedione to estrone, the reaction mediated by the aromatase enzyme complex, may make an important contribution to the synthesis of estrogens in breast tissues. In the present study, the effect of the cytokine, TNFα, on aromatase activity was examined in breast fibroblasts derived from normal and malignant breast tissue. TNFα (2.5–10.0 ng/ml), in the presence of stripped fetal calf serum and dexamethasone, significantly stimulated fibroblast aromatase activity in a dose-dependent manner. IL-1 and IL-6 also stimulated fibroblast aromatase activity, but no marked synergism between TNFα and IL-1 or IL-6 was detected. Using a specific radioimmunoassay, significant concentrations of TNFα were detected in samples of breast cyst fluid and breast tumor cytosol, which had previously been shown to stimulate aromatase activity, but not in conditioned medium from breast tumor-derived fibroblasts. As TNFα may be preferentially expressed and produced in the adipose tissue component of the breast, this cytokine may have an important role in regulating estrogen synthesis in normal and malignant breast tissues.
The interaction of cytokines in regulating oestradiol 17β-hydroxysteroid dehydrogenase activity in MCF-7 cells
1994, Journal of Steroid Biochemistry and Molecular BiologyOestradiol 17γ-hydroxysteroid dehydrogenase (E2DH) has a pivotal role in the regulation of oestradiol (E2) concentrations in normal and amlignant breast tissues. previous studies have suggested that a number of cytokines can stimulate E2DH activity to increase the conversion of oestrone (E1 to E2. In this investigation we have examined the effect of TNFα, interleukin-1β (IL-1β) and IL-6 on E2DH activity in MCF-7 breast cancer cells. These cytokines may produced by breast tumours and their presence in conditioned medium (CM) from tumour-derived fibroblasts was also measured to assess their possible contribution to its E2DH stimulatory activity. Treatment of MCF-7 cells with IL-1β and TNFα (5 ng/ml) significantly increased (P < 0.001) reductive E2DH (red-E2DH, the conversion of E1 to E2) activity. In contrast, IL-6 at a concentration of 100 ng/ml produced little, if any, stimulation of reductive activity. Combinations of all three cytokines acted synergistically to stimulate red-E2DH activity. No cytokine, either alone or in combination, affected oxidative () activity. Significant concentrations of IL-6 and IL-1β were detected in CM, but the stimulation of red-E2DH activity was much greater than that which could be explained by their levels alone. It is concluded that these cytokines may play an important role in regulating E2DH activity in breast cancer cells and may act synergistically in vivo to enhance the formation of E2 in breast tumours.