Effect of treatment with 4-hydroxyandrostenedione on the peripheral conversion of androstenedione to estrone and in vitro tumor aromatase activity in postmenopausal women with breast cancer.

The effect of treatment with the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) on the peripheral conversion of androstenedione to estrone has been examined in eight postmenopausal women with advanced breast cancer. Before treatment conversion of androstenedione to estrone ([p]AEIBB) ranged from 0.81 to 3.7% and was almost completely inhibited after treatment with 4-OHA (two doses of 500 mg i.m. with an interval of 12 days between doses). Transfer constants were also measured by the urinary method ([p]AEIBU) for some subjects and decreased from 2.3 +/- 0.52% to 0.24 +/- 0.11% after treatment, a mean reduction of 90%. Mean plasma concentration of estradiol (37.4 +/- 16.6 pmol/liter) and estrone (99.0 +/- 32.2 pmol/liter) decreased significantly (P less than 0.01) to 15.7 +/- 4.6 pmol/liter and 52.4 +/- 8.9 pmol/liter, respectively, after treatment. Aromatase and DNA polymerase alpha (a marker of cell proliferation) activities were measured in seven samples of breast tumor tissue obtained before and after treatment. For three samples there was a marked (67 +/- 17%) decrease in tumor aromatase activity after treatment, for two, little change occurred, while tumor aromatase activity in the other two samples appeared to be resistant to the effect of 4-OHA. The correlation between tumor aromatase and DNA polymerase alpha activities (r = 0.45) failed to reach a significant level.

Characterization of neutral TRH-like peptides in mammary gland, mammary tumors and milk.

Three pyroglutamylpeptide amides, pGlu-Glu-Pro amide, pGlu-Phe-Pro amide and pGlu-Gln-Pro amide, with similar structures to thyrotropin-releasing hormone (TRH), have been identified previously in the male reproductive system. We report here that rat and human mammary gland contain neutral TRH-immunoreactive peptides which are not retained on cation or anion exchange chromatography and that similar peptides occur in the milk of rat, cow, ewe and sow. The TRH-like peptides in lyophilized milk from the cow were purified by gel exclusion chromatography, mini-column cation exchange chromatography and reversed phase high performance liquid chromatography (HPLC) and the chromatographed peptides were located by TRH radioimmunoassay (RIA). In each chromatographic system the major TRH-immunoreactive peptide from cow milk exhibited identical behavior to pGlu-Phe-Pro amide; in addition there were two minor TRH-immunoreactive components. The possible physiological role of the TRH-like peptides in the mammary gland is discussed. In a series of patients with breast carcinoma, mammary tumor tissue was shown to contain approximately four times more TRH-like peptide than normal mammary tissue from the same patient, raising the possibility that the TRH-like peptides may be implicated in tumor development.

Regulation of aromatase activity by cytokines, PGE2 and 2-methoxyoestrone-3-O-sulphamate in fibroblasts derived from normal and malignant breast tissues.

Synthesis of oestrone from androstenedione within tumours, by the aromatase enzyme complex, is an important source of oestrogen that is available to support the growth of hormone-dependent breast tumours. In view of the central role that the aromatase enzyme has in oestrogen synthesis there has been considerable interest in understanding its regulation and developing inhibitors to block its action. In the present study we have derived fibroblasts from breast tumours (TFs), tissue proximal to tumours (PFs) and reduction mammoplasty tissue (RMFs) and used them to investigate the regulation of aromatase activity by PGE(2), IL-6 plus its soluble receptor (SR) or TNFalpha. In addition we have examined the ability of 2-methoxyoestrone sulphamate (2-MeOEMATE), a compound which alters microtubule stability, to block the stimulation of aromatase activity by these factors. Basal aromatase activity in PFs was significantly higher (p<0.001) than in TFs or RMFs. The combination of IL-6 plus SR or TNFalpha produced the greatest stimulation of aromatase activity in TFs (up to 61-fold) while having a much lower stimulatory effects on aromatase activity in PFs (up to 60% increase) or RMFs (up to 192% increase). 2-MeOEMATE reduced basal aromatase activity in TFs by 87% and completely abrogated the ability of PGE(2), IL-6 plus SR or TNFalpha to stimulate aromatase activity in these fibroblasts. Results from these studies indicate that while PFs have the highest level of non-stimulated aromatase activity, aromatase activity in TFs shows the greatest response to cytokines. These findings suggest that intrinsic difference may exist for the different types of fibroblasts in the way in which they respond to regulatory factors. The ability of 2-MeOEMATE to block cytokine stimulated aromatase activity suggests that, in addition to its other anti-cancer properties, this compound may also act to inhibit cytokine-stimulated aromatase activity in breast tumours.

The regulation of aromatase activity in breast fibroblasts: the role of interleukin-6 and prostaglandin E2.

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.

Aromatase activity and interleukin-6 production by normal and malignant breast tissues.

The aromatase enzyme complex, which regulates the conversion of androstenedione to estrone, may have an important role in regulating estrogen synthesis in breast tissues. In this study the effect of tumor location on aromatase activity in adjacent tissue was examined and related to interleukin-6 (IL-6) production, which has been shown to stimulate aromatase activity in breast cancer cells. Samples of normal and malignant breast tissues were obtained from 11 women undergoing mastectomy. In 7 patients, aromatase activity was highest in the quadrant in which the tumor was located or on which the tumor impinged. Aromatase activity in tumor-bearing quadrants was significantly higher than that in adjacent and opposite quadrants. Aromatase activity and IL-6 production, expressed in terms of tissue weight, were significantly higher for tumor tissue compared with normal breast adipose tissue. A significant correlation was found between aromatase activity and IL-6 production for breast tumor tissue (rs = 0.56; P < 0.05), but not for adipose tissue from the breast quadrants. Aromatase activity and IL-6 production were also measured in tissue obtained from a normal woman undergoing reduction mammoplasty who had previously had breast augmentation by silicone injection, not contained within a capsule. In tissue from this patient there was evidence of chronic inflammation and a marked macrophage response. Aromatase activity in this tissue was considerably higher than that detected in mastectomy adipose tissue samples, and a significant correlation was found between aromatase activity and IL-6 production (rs = 0.77; P < 0.05). A preliminary study to examine the potential role of cells of the immune system in regulating breast tissue aromatase activity revealed that conditioned medium collected from macrophages and lymphocytes could markedly stimulate aromatase activity in tumor-derived fibroblasts. The results of this study confirmed that breast tumor location can influence aromatase activity in adjacent tissues and showed that aromatase activity is increased in tumor-bearing quadrants. The increased production of IL-6 by tumor tissue and its correlation with aromatase activity suggest that tumors may be the major source of IL-6, which is able to influence aromatase activity in adjacent tissues.

Epidermal growth factor and oestradiol in human breast cyst fluid.

Gross cystic breast disease is a common condition in women. Women with apocrine breast cysts (breast cyst fluid Na+/K+ less than 3) may be at higher risk of breast cancer than women who have cysts lined by flattened epithelium (Na+/K+ greater than or equal to 3). Breast cyst fluid concentrations of epidermal growth factor were significantly higher in the low electrolyte ratio group than in the high electrolyte ratio group (356.2 ng/ml vs 104.1 ng/ml, P less than 0.0003). A negative correlation was obtained between intracystic epidermal growth factor concentrations and Na+/K+ (rs = -0.666, P less than 0.001). No significant difference was found between the total oestradiol concentrations in the two cyst groups. However, the unbound oestradiol concentrations on a limited number of samples were significantly higher in the low electrolyte ratio group than in the high electrolyte ratio group (P less than 0.05). The higher concentrations of EGF in cyst fluid with Na+K+ less than 3 may explain why women with apocrine breast cysts may be at increased risk of developing breast cancer.

The effect of breast tumour and normal breast tissue cytosols on oestradiol 17 beta-hydroxysteroid dehydrogenase activity.

We have prepared cytosols of normal breast tissue and breast tumour tissue and examined their effect on 17 beta-oestradiol dehydrogenase (E2DH) activity in cultured normal breast fibroblasts and on the MCF-7 breast cancer cell line. Tumour cytosol increased the activity of E2DH in the reductive (E1----E2) direction in both MCF-7 cells and normal breast fibroblasts. E2DH activity in the oxidative (E2----E1) direction did not change. Increasing the amount of tumour cytosol progressively raised E2DH activity in the E1----E2 direction in MCF-7 cells, whereas equivalent amounts of cytosol from normal tissue had no effect. No change was seen in the oxidative direction. These findings suggest that a factor present in breast tumours may influence E2DH activity and consequently, the hormonal environment and growth of tumours.

The effect of epidermal growth factor, transforming growth factor and breast tumour homogenates on the activity of oestradiol 17 beta hydroxysteroid dehydrogenase in cultured adipose tissue.

The effect of epidermal growth factor (EGF), transforming growth factor (TGF alpha) and breast tumour homogenates on oestradiol 17 beta hydroxysteroid dehydrogenase (E2DH) activity has been examined using cultured human breast adipose tissue. EGF (100-1000 ng/ml) inhibited E2DH activity (E1----E2) in a dose dependent manner. TGF alpha (250 and 500 ng/ml) stimulated E2DH activity, with conversion of E1----E2 increasing to a greater degree than E2----E1 activity. Breast tumour homogenates (2-10% w/v) also influenced E2DH activity. It is concluded that growth factors, produced by breast tumours, may modulate E2DH activity in tissues surrounding the tumour and thereby influence tumour growth.

Effect of breast cyst fluid on oestrogen 17-oxidoreductase activity in MCF-7 breast cancer cells.

Breast cyst fluid (BCF) was found to stimulate oestrogen 17-oxidoreductase activity in the reductive direction, i.e., conversion of oestrone (E1) to oestradiol (E2), in MCF-7 breast cancer cells. Dialysis of BCF revealed that this property of BCF was present in both dialysed BCF and dialysate, implying that both high and low mol. wt. substances were responsible for stimulating E1 to E2 conversion. Gel filtration of dialysed BCF revealed that the high mol. wt. substances responsible for the stimulation of E1 to E2 conversion had mol. wts. of approximately 11 kD and 68 kD. This property of BCF would serve to increase the concentration of E2, a steroid which may play a role in mammary carcinogenesis.

Regulation of aromatase and sulphatase in breast tumour cells.

In situ oestrogen synthesis makes an important contribution to the high oestrogen concentration found in breast tumours. Cytokines, including interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha), have been shown to regulate aromatase activity in fibroblasts derived from normal and malignant breast tissues. In the present study, the ability of other cytokines in the IL-6 superfamily (IL-11 and oncostatin M) to stimulate aromatase activity has been confirmed. Formation of oestrone via the oestrone sulphatase pathway may be the major route of tumour oestrogen synthesis and in the present study TNF alpha was found to stimulate sulphatase activity in a dose-dependent manner. Human serum albumin was also found to be a potent stimulator of oestrone sulphatase activity. Its stimulatory effect was blocked by basic fibroblast growth factor, but not by several other growth factors tested. Insight into the regulation of oestrogen synthesis in breast tumours should enable the development of novel compounds to inhibit oestrogen synthesis in women with breast cancer.

IL-6sR: release from MCF-7 breast cancer cells and role in regulating peripheral oestrogen synthesis.

The interleukin-6 soluble receptor (IL-6sR) may regulate the ability of IL-6 to stimulate oestrogen synthesis in breast cancer cells and breast tumours. Significant aromatase activity was detectable in IL-6 stimulated fibroblasts derived from subcutaneous adipose tissue, but the combination of IL-6sR plus IL-6 resulted in a marked 21-fold stimulation of aromatase activity. To examine the control of IL-6sR release, the effects of oestradiol, 4-hydroxytamoxifen (4-OHT), dexamethasone, TPA, TNF alpha or IL-6 on this process was examined using MCF-7 breast cancer cells. Oestradiol, TNF alpha and dexamethasone all markedly increased IL-6sR release. While 4-OHT had a small stimulatory effect on IL-6sR release, it blocked the ability of oestradiol to increase IL-6sR release. Significant concentrations of IL-6sR were also detected in conditioned medium collected from lymphocytes and macrophages and in cytosols prepared from normal and malignant breast tissues. These results indicate that IL-6sR may have an important role in potentiating the effect of IL-6 on oestrogen synthesis in breast cancer cells. The abilities of oestradiol or tamoxifen to potentiate or inhibit the IL-6 stimulation of oestrogen synthesis in breast cancer cells may result from their effects on IL-6sR release.

Interleukin-1 and interleukin-6 in breast cyst fluid: their role in regulating aromatase activity in breast cancer cells.

Gross cystic breast disease is a common benign disease which may be associated with an increased risk for breast cancer. Breast cyst fluid (BCF) contains many steroids, peptide growth factors and proteins. We have now identified interleukin-1 (IL-1) and IL-6 in BCF by specific radioimmunoassays. Concentrations of IL-1 were similar in BCF with low or high Na+/K+ ratios (ratio less than 3 vs greater than 3; 357 +/- 72 pg/ml vs 308 +/- 126 pg/ml). In contrast, IL-6 concentrations were significantly higher (P less than 0.01) in BCF with a Na+/K+ ratio greater than 3 (2.75 +/- 2.34 ng/ml) compared with BCF with a low electrolyte ratio (0.21 +/- 0.09 ng/ml). BCF (10%, v/v) stimulated aromatase activity when added to dexamethasone stimulated breast tumour-derived fibroblasts and there was a significant correlation between the stimulation of aromatase activity and BCF Na+/K+ ratio (r = 0.95, P less than 0.001). A significant correlation was also found between stimulation of aromatase activity and concentration of IL-6 in BCF (r = 0.80, P less than 0.01) but not IL-1 concentration (r = -0.39, not significant). Addition of IL-1 or IL-6 (50 ng/ml) to fibroblasts stimulated aromatase activity but was associated with a small (20%) decrease in cell growth. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast cancer cells.

Identification of albumin in breast tumor cytosol as a factor involved in the stimulation of estradiol 17 beta-hydroxysteroid dehydrogenase (reductive) activity.

Estradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) is the enzyme responsible for the interconversion of estrone (E1), and the more biologically potent steroid, estradiol (E2), and has a crucial role in regulating breast tissue concentrations of E2. It has previously been shown that breast tumor cytosol is able to preferentially stimulate the reductive conversion of E1 to E2 in cultured MCF-7 breast cancer cells. In this study the stimulatory factor(s) from breast tumor cytosol have been partially purified by gel filtration and affinity chromatography. Human serum albumin (HSA) has been identified as a component of this bioactive fraction. Subsequent testing of commercially purified HSA preparations has revealed the ability of some preparations to be highly stimulatory. The albumin present in breast tumor cytosol may therefore be a contributing factor to the observed stimulation of reductive E2DH activity in cultured MCF-7 cells. Such a mechanism may account in part for the higher concentrations of E2 which are observed in breast tumors in vivo.

Regulation of oestradiol 17 beta hydroxysteroid dehydrogenase in breast tissues: the role of growth factors.

Oestradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) is present in normal and malignant breast tissues and regulates the interconversion of oestrone and the biologically active oestrogen, oestradiol. Studies we have previously carried out have indicated that concentrations of oestradiol and the conversion of oestrone to oestradiol are higher in breast tumours than in normal breast tissues. We are currently isolating and characterizing factors produced by breast tumours which are capable of stimulating E2DH (reductive) activity. The production of such factors by breast tumours, which stimulate the conversion of oestrone to oestradiol, would provide a favourable oestrogenic environment to promote tumour growth and may account for the increased concentrations of oestradiol in breast tumours.

Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors.

The aromatase complex has a key role in regulating oestrogen formation in normal and malignant breast tissues. Using dexamethasone-treated fibroblasts, derived from breast tumours, breast tumour cytosol and breast tumour-derived conditioned medium (CM) markedly stimulate aromatase activity. The cytokine, interleukin-6 (IL-6) has been identified as a factor present in CM which is capable of stimulating aromatase activity. To examine whether IL-6 may have a role in vivo in regulating breast tissue aromatase activity, IL-6 production and aromatase activity in breast tumour and adipose tissue from breast quadrants were examined. In 5/6 breasts examined so far, aromatase activity was highest in adipose tissue in the breast quadrant containing the tumour or on which the tumour impinged. There was a significant correlation (P < 0.05, Kendall's rank correlation) between IL-6 production and aromatase activity in these breast tissues. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast tissues.

Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts.

The cytokine interleukin-6 (IL-6) and its soluble receptor (IL-6sR) can act synergistically to stimulate aromatase activity in cultured stromal fibroblasts derived from breast tissues. In this study, a 16 amino acid peptide, AROHIB, has been used in an attempt to block the ability of IL-6 plus IL-6sR to stimulate aromatase activity in stromal fibroblasts. Pre-incubation of cells with AROHIB for a 3-h period before the addition of IL-6 and IL-6sR resulted in a marked (67%) reduction in the ability of these factors to stimulate aromatase activity. AROHIB was found to be rapidly degraded when exposed to MCF-7 breast cancer cells or fibroblasts. Analysis by FAB-MS was used to identify the site of peptide cleavage. Subsequently, a series of 10 amino acid peptides, DP1-DP4, were designed, synthesised and tested for their ability to resist proteolytic degradation and to inhibit IL-6 plus IL-6sR-stimulated aromatase activity. Peptide DP2, a modified version of the active fragment of AROHIB, had N-acetyl and C-amino terminal protection and an internal D-amino acid (instead of L form) at the site of proteolytic cleavage. Using cells cultured in the presence of 2% stripped foetal calf serum, peptide DP2 resulted in a 74% reduction in cytokine-stimulated aromatase activity. Under serum-free conditions, peptides DP1-DP3 showed modest inhibitory properties. Results from this study suggest that it may be possible to develop small peptides to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner.

Regulation of steroid sulphatase expression and activity in breast cancer.

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.

Control of aromatase activity in breast tumours: the role of the immune system.

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF alpha has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.

In vivo conversion of norethisterone to ethynyloestradiol in perimenopausal women.

The extent to which norethisterone is converted to ethynyloestradiol is controversial. To investigate the conversion of norethisterone to ethynyloestradiol we have used a double isotope infusion technique to measure the conversion in vivo. The use of acids or bases was precluded to prevent possible artefactual formation of phenolic metabolites of norethisterone. Transfer constants for the conversion of norethisterone to ethynyloestradiol in two perimenopausal women were 2.26 and 2.34% as measured in blood and 2.27 and 0.38% in urine. Results from this study show that a small but significant proportion of norethisterone is converted to ethynyloestradiol in vivo.

Conversion of estrone to estradiol and estradiol to estrone in postmenopausal women.

The conversion of estrone to estradiol and metabolism of estradiol to estrone have been measured in postmenopausal women to examine factors that influence these conversions. Transfer constants for the conversion of estrone to estradiol and estradiol to estrone were not significantly correlated with age (r = -.01 and r = .1, respectively) or subjects' percentage of ideal body weight (r = -.25 and r = -.22, respectively). There was a significant negative correlation between the transfer constants for the conversion of estradiol to estrone and plasma levels of dehydroepiandrosterone sulphate (r = -.45, P less than .05) but not plasma levels of progesterone. Transfer constants for the conversion of estrone to estradiol and metabolism of estradiol to estrone in women with breast or endometrial disease were similar to values in normal women, but metabolism of estradiol to estrone was elevated in two women with primary biliary cirrhosis.