Estrogen and progestin bioactivity of foods, herbs, and spices.

In this study we report on the content and bioactivity of plant (phyto) estrogens and progestins in various foods, herbs, and spices, before and after human consumption. Over 150 herbs traditionally used by herbalists for treating a variety of health problems were extracted and tested for their relative capacity to compete with estradiol and progesterone binding to intracellular receptors for progesterone (PR) and estradiol (ER) in intact human breast cancer cells. The six highest ER-binding herbs that are commonly consumed were soy, licorice, red clover, thyme, tumeric, hops, and verbena. The six highest PR-binding herbs and spices commonly consumed were oregano, verbena, tumeric, thyme, red clover and damiana. Some of the herbs and spices found to contain high phytoestrogens and phytoprogestins were further tested for bioactivity based on their ability to regulate cell growth rate in ER (+) and ER (-) breast cancer cell lines and to induce or inhibit the synthesis of alkaline phosphatase, an end product of progesterone action, in PR (+) cells. In general, we found that ER-binding herbal extracts were agonists, much like estradiol, whereas PR-binding extracts, were neutral or antagonists. The bioavailability of phytoestrogens and phytoprogestins in vivo were studied by quantitating the ER-binding and PR-binding capacity of saliva following consumption of soy milk, exogenous progesterone, medroxyprogesterone acetate, or wild mexican yam products containing diosgenin. Soy milk caused a dramatic increase in saliva ER-binding components without a concomitant rise in estradiol. Consumption of PR-binding herbs increased the progestin activity of saliva, but there were marked differences in bioactivity. In summary, we have demonstrated that many of the commonly consumed foods, herbs, and spices contain phytoestrogens and phytoprogestins that act as agonists and antagonists in vivo.

Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture.

We investigated the estrogenic activity of various environmental pollutants (xenobiotics), in particular the xenoestrogen o,p-DDT, and compared their effects with those of endogenous estrogens, phytoestrogens, and mycoestrogens on estrogen receptor binding capacity, induction of estrogen end products, and activation of cell proliferation in estrogen-sensitive human breast cancer cells in monolayer culture. We also quantified the levels of phytoestrogens in extracts of some common foods, herbs, and spices and in human saliva following consumption of a high phytoestrogen food source (soy milk) to compare phytoestrogen abundance and bioavailability relative to the reported xenoestrogen burden in humans. Results show that natural endogenous estrogens, phytoestrogens, mycoestrogens, and xenoestrogens bind estrogen receptor (ER) in intact cells, but demonstrate marked differences in their ability to induce end products of estrogen action and to regulate cell proliferation. All of the different classes of estrogens stimulated cell proliferation at concentrations that half-saturated ER, but only some classes were able to induce estrogen-regulated end products. Genistein, a common phytoestrogen found in soy foods, differed from the xenoestrogen DDT in its effects on cell proliferation and ability to induce estrogen-regulated end products. Moreover, we found that many of the foods, herbs, and spices commonly consumed by humans contain significant amounts of phytoestrogens, and consumption of soy milk, a phytoestrogen-rich food, markedly increases the levels of phytoestrogens in saliva. In conclusion, our in vitro results predict that a diet high in phytoestrogens would significantly reduce the binding of weak xenoestrogens to ER in target tissues in vivo.

Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro.

Experimental and epidemiologic studies support the view that soyfoods prevent cancer as well as diseases and symptoms associated with estrogen deficiency. Recent research suggests that the isoflavonoid genistein, a phytoestrogen found in abundance in soyfoods, may be one of the principal molecular components responsible for these health benefits. In this study we investigated the effects of a broad physiologically relevant concentration range of genistein on estrogen receptor (ER) binding, induction of the estrogen-regulated antigen pS2, and cell proliferation rate in ER(+) and ER(-) human breast cancer cells grown in vitro. Dose response to genistein was compared with that of estradiol, tamoxifen, and several other structurally similar iso- and bioflavonoids (e.g., equol, kaempferol, and quercetin). Our results revealed that genistein has potent estrogen agonist and cell growth-inhibitory actions over a physiologically achievable concentration range (10 nM-20 microM). Other flavonoids over the same concentration range were good estrogen agonists and poor cell growth inhibitors (equol) or poor estrogen agonists and potent growth inhibitors (kaempferol and quercetin). The growth-inhibitory actions of flavonoids were distinctly different from those of triphenyl antiestrogens like tamoxifen. In summary, our results reveal that genistein is unique among the flavonoids tested, in that it has potent estrogen agonist and cell growth-inhibitory actions over a physiologically relevant concentration range.

Prognostic value of Cathepsin D expression in breast cancer: immunohistochemical assessment and correlation with radiometric assay.

BACKGROUND: The prognostic significance of Cathepsin D and optimal methodologies to measure Cathepsin D in breast cancers are controversial. PATIENTS AND METHODS: Quantitative (immunoradiometric) and semiquantitative (immunohistochemical) assays for Cathepsin D expression were compared using 25 breast carcinomas. Immunohistochemical Cathepsin D results were derived using 3 different anti-Cathepsin D antibodies and significant associations between immunohistochemical and radiometric Cathepsin D data were observed for each reagent. Immunohistochemical analysis of Cathepsin D expression was performed on nearly 500 fixed-embedded archival breast cancers with long-term patient follow-up using 2 anti-Cathepsin D antibodies (CDR2-11/23, IC11). RESULTS: The immunohistochemical reagents recognized generally overlapping subsets of Cathepsin D positive tumors (correlation co-efficient 0.54; p = 0.00016). Correlations between Cathepsin D data and clinical, histologic or biologic features differed for each antibody. For the node-negative patient subset, Cathepsin D immunopositivity correlated with erbB-2 and stress-response protein 27 overexpression but not survival. Cathepsin D positivity was associated with subsequent distant metastasis and estrogen receptor positivity in node positive patients. Univariate analysis of all patients suggested that Cathepsin D immunopositivity may be predictive of a reduced metastasis-free but not overall survival. Multivariate analysis, however, failed to confirm an independent prognostic value for Cathepsin D in breast cancer patients. CONCLUSIONS: These data do not confirm an independent prognostic significance for Cathepsin D using immunohistochemical methods on breast cancers.

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.

Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule.

The preparation of monoclonal antibodies (MAbs) against the human milk fat globule membrane with preferential binding to breast carcinoma cells is described. Using BALB/c mouse myeloma cells; inter-specific, intra-strain, and inter-strain hybridomas were isolated that identified three different components of the human milk fat globule of approximately 46,000, and 70,000 daltons and a mucin-like glycoprotein complex (NPGP) ranging from 400,000 to over a million daltons, respectively. Three MAbs (BrE1, BrE2, BrE3) identified the latter component which consists of at least three different size molecules for which the aforementioned MAb's have different binding specificities. MAbs, BrE2 and BrE3, bound to normal breast epithelial cells but to a lesser extent than to tumors and only at the apical surface facing the lumen, while they bound breast carcinomas strongly, and often in the cytoplasm as well as on the surface. Higher concentrations of BrE3 were required to stain normal breast compared to breast tumors. BrE1 also stained breast carcinomas both on the surface and cytoplasmically but did not stain normal breast tissue. The MAb, Mc13, as well as the previously reported MAb McR2, both against the 70,000 dalton component, did not significantly stain either normal or cancerous breast tissue in histological sections but did bind significantly to cultured breast epithelial cells and to the milk fat globule membrane. The MAbs, Mc8 and Mc3, reported previously to be against the 46,000 dalton component, stained histologically only malignant breast tissue but only weakly; however, they bound strongly to intact breast carcinoma cells and breast cell membrane preparations with a radioimmunobinding assay. These MAbs should be useful in characterizing the surface of breast epithelial cells, studying surface alterations in malignancy, and possibly in breast cancer diagnosis and therapy.

Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies.

Monoclonal antibodies (MAbs), recently produced against human progesterone receptors (PR), were used for immunocytochemical localization of PR. The specificity of the immunocytochemical assay for PR was demonstrated by incubation with control MAbs, preabsorption of MAbs with highly purified human PR, and by the cell and tissue distribution of the immunostaining reaction. With human breast cancer cell lines, immunoreactivity was confined to cells that contain PR by steroid-binding assay. Moreover, immunostaining was induced by estradiol in estrogen-responsive cells, MCF-7 and ZR-75-1. In a preliminary study with 33 breast carcinomas, a good correspondence was obtained between immunocytochemical staining and PR content assessed by conventional steroid-binding assay. Immunoperoxidase localization was also obtained with other human target tissues. In normal breast and benign breast disease, immunoreactivity was observed with nuclei of ductal epithelial cells and hyperplastic epithelium. In uterus, immunostaining of endometrium was localized to nuclei of stromal and glandular epithelial cells and in myometrium to nuclei of smooth muscle cells. The effect of the progestin agonist, R5020, and antagonist, RU 486, on PR localization was investigated with the PR-rich T47D human breast cancer cell line. In the absence of hormone, immunostaining was exclusively nuclear. This was true under a number of cell culture conditions designed to eliminate endogenous progestins from the culture medium. Exclusive nuclear localization of PR was not due to a failure of the MAbs to recognize unoccupied PR, since each MAb bound equally well in vitro with different receptor forms. These included liganded and unliganded cytosol PR, molybdate stabilized PR, and nuclear-transformed receptors. Nor was failure to detect cytoplasmic staining due to a selective destruction or loss of unoccupied PR from the cytoplasmic compartment as a result of cell fixation. This was assessed by dot blot immunoassay of PR antigen distribution in subcellular fractions of fixed and unfixed cells. Continuous exposure of cells to R5020 resulted in a transient (30-60 min) increase in nuclear staining intensity (without change in cytoplasmic reactivity), followed by a progressive decline in immunoreactivity. By 24 h of R5020 treatment, the vast majority of cells displayed no immunostaining reaction. These immunocytochemical data are consistent with progestins down regulating their own receptors due to a loss in cellular PR content and not to inactivation of receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer.

A monoclonal antibody (323/A3) with a high degree of selectivity for binding to breast cancer cells was produced by immunization of mice with MCF-7 human breast cancer cells. The antigen recognized by 323/A3 on MCF-7 appears to be surface localized, and by enzyme-linked immunosorbent assay, the antibody was found to bind strongly with four of six breast cancer cell lines examined while no binding was detectable with nonbreast cancer cell lines. In vivo distribution of the 323/A3 antigen was screened by immunoperoxidase staining of formalin-fixed paraffin sections of normal human tissues and tumors. Among breast tissues, positive staining was detected with 75% (6 of 8) of metastatic lymph nodes, 59% (76 of 128) of primary breast tumors, 20% (13 of 63) of benign breast lesions, and 0% (0 of 10) of normal breast. No immunostaining was detected with a large variety and number of other normal human tissues with the exception of staining observed with epithelium of normal colon. Antigen distribution appears not to be disease specific, since positive staining was also observed with adenocarcinomas other than breast. The antigen recognized by the 323/A3 antibody was identified by Western blot analysis as a Mr 43,000 protein. The glycoprotein nature of the antigen was demonstrated by its binding to concanavalin A, specific elution with sugar, and immunoprecipitation of a Mr 43,000 radiolabeled protein from extracts of MCF-7 cells after pulse labeling with [3H]glucosamine. The 323/A3 antigen appears to be the same Mr 43,000 protein in cell lines as in breast tumors in vivo. Based on a comparison with the molecular weights of other known tumor-associated antigens and with their immunocytochemical tissue distribution, the Mr 43,000 glycoprotein described here represents a tumor-associated antigen previously undescribed in breast cancer or in other tumors. Since the Mr 43,000 glycoprotein is present on the surface of most breast cancer cells and is either absent or expressed at very low levels in most normal tissues including normal breast, the monoclonal antibody described here may have potential applications in diagnosis and management of breast cancer.

Antiestrogenic therapy of meningiomas--a pilot study.

Six patients with inoperable, nonoperative, or recurrent meningiomas were treated with the antiestrogenic agent tamoxifen (Nolvadex) during an 8-12-month period. Computer tomographic, scintigraphic, and clinical evidence of an unspecific tumor response was only encountered in one patient after 4 months of therapy with tamoxifen. The 2-year results did not indicate a favorable response to antiestrogenic treatment. The significance of sex-steroid receptors and their possible prognostic value in endocrine therapy of meningiomas is discussed.

[Intratumoral regional differences in hormone receptor status of breast cancer]. / Intratumorale regionäre Unterschiede im Hormonrezeptor-Status des Mammakarzinoms.

Biopsy specimens of tissue were taken from 32 invasive carcinomas of the breast, in each case from the centre of the tumour, the tumour margin and the surrounding mammary gland tissue. After preparation of a frozen section for tissue identification, the concentration of oestrogen and progesteron receptors (ER and PR, respectively) was determined in each specimen by means of the dextrane-coated charcoal method. On the basis of these measured values, the receptor status of each specimen was classified either as positive (R+), borderline value (BL) or as negative (R-). Various morphological parameters were compared with the measured data. 9 (28%) of the 32 carcinomas demonstrated such high intratumoural differences in receptor concentration that the receptor status was classified quite differently in each of them. Formation of tubuli of the invasive ductal carcinomas was more marked with the R+ tumours (p = 0.005) than with the R- tumours. The quantitative ER content did not correlate with the abundance of the tissue specimen (r = 0.182). The regional differences in receptor status could not be explained, neither by the abundance in cells nor by morphological peculiarities of the tumour. - It follows from the results of this study that intratumoural regional variations in ER and PR concentrations can occur in carcinoma of the breast, and that such variations can have clinical significance.

A simple method to determine whole cell uptake of radiolabelled oestrogen and progesterone and their subcellular localization in breast cancer cell lines in monolayer culture.

Specific uptake of tritiated 17 beta-oestradiol and R5020, a synthetic progestin, in breast cancer cell lines ( MCF7 and T47D) growing in monolayer culture in multiwell plates has been shown. Binding characteristics, calculated by Scatchard analysis, indicate the presence of steroid receptors of similar affinities and capacities to those already obtained with broken cell preparations. Lysis of the cells by treatment with a hypotonic buffer reveals the subcellular localization of the receptors so the method can be used to study receptor dynamics such as nuclear translocation and processing. Cell growth can be measured by DNA determination directly in the multiwell plates. Thus, the method provides a convenient way of studying the effects of steroid hormones (or any antihormone or chemotherapeutic agent) on growth and receptor content of breast cancer cells in monolayer culture.

Estrogen receptor-mediated and cytotoxic effects of the antiestrogens tamoxifen and 4-hydroxytamoxifen.

The triphenylethylene antiestrogen tamoxifen (TAM) is believed to exert its antitumor effect via the estrogen receptor (ER). To test this hypothesis and to differentiate between ER-mediated and general cytotoxic effects of TAM, the growth-inhibitory effects of TAM and its in vivo metabolite 4-hydroxytamoxifen (OH-TAM) have been studied in five continuous human cancer cell lines, MCF7 and T47D (mammary carcinoma, ER positive), BT20 and MDA-MB-231 (mammary carcinoma, ER negative), and ME8 (melanoma, ER negative). All five cell lines are completely killed by concentrations of TAM and OH-TAM above 10(-6) M, regardless of ER status. TAM and OH-TAM have little effect on the ER-negative lines at concentrations below 10(-6) M, whereas the ER-positive lines are highly sensitive to TAM at 10(-7) M and to OH-TAM at 10(-9) M. Inhibition of growth parallels the relative affinity of these drugs for the ER. We conclude that, above 10(-6) M, the growth-inhibitory effects of TAM and OH-TAM in tissue culture are the results of a mechanism other than that via the ER system and that only at lower concentrations are the true ER-mediated effects seen. Plasma concentrations of TAM and OH-TAM in breast cancer patients treated with TAM are in the same range as the concentrations in vivo at which growth inhibition is seen, leading to the conclusion that both compounds contribute to the overall effect of TAM in vivo.

Receptor heterogeneity of human breast cancer as measured by multiple intratumoral assays of estrogen and progesterone receptor.

Multiple intratumoral tissue samples from the primary mass of 30 consecutive invasive breast cancer patients were assayed for estrogen receptor (ER) and progesterone receptor (PR) by the dextran-coated charcoal method following frozen section histopathological examination. Steroid receptor status of each sample was classified as positive (R+) or negative (R-), based only upon quantitative guide lines from the ER/PR results. Four out of 32 (12.5%) of the invasive cancers had an intratumoral sample classified as R+ and one sample as R-. R+ invasive ductal carcinomas has a highly significant degree of tubule formation (P = 0.005) when compared with R- invasive ductal cancers. While the quantitative ER content (r = 0.18) and the degree of quantitative variation in ER content (P = 0.04) did not correlate with the tumor cellularity of the individual samples, tumor cellularity (P = 0.005) and ER content (P = 0.005) were lower in the samples from the tumor border than from the central tumor samples. Variations in ER and PR content may be found on a regional basis within a breast tumor mass resulting from heterogeneity of tumor subpopulations and/or differences in tumor cellularity.

Estrogen and progestin receptors in meningiomas: clinicopathological correlations.

Estradiol and progesterone receptors were studied in 44 patients with meningiomas and correlated to the clinicopathological features and amount of preoperative corticosteroid therapy. Thirty-four (77%) of the meningiomas contained high titers of specific high-affinity cytosol [3H]promegestone (R 5020) binding sites (mean 2,902 fmol/g tumor; range 0-9,598 fmol/g tumor) whereas only miniscule amounts of a nonspecific cytoplasmic [3H]estradiol binding component (mean 48 fmol/g tumor; range 0-201 fmol/g tumor) were detectable. No nuclear binding activity for [3H]estradiol was demonstrable. There was no convincing correlation between high PR activity and the age, sex, or menopausal status of the patients. The correlation study between the amount of preoperative corticosteroid therapy with the amount of [3H]promegestone binding revealed no dose relationship. Correlating [3H]promegestone content with the histologic type, we found 96% of meningothelial, 71% of transitional, and 40% of fibroplastic meningiomas to contain progesterone receptors. The necessity of in vitro studies is stressed to assess the biosynthesis and biological activity of the progesterone receptor in meningiomas, which is apparently not estrogen regulated, as is the case in other estrogen target tissues.

Biological expression of steroid hormone receptors in primary meningioma cells in monolayer culture.

Primary meningiomas have been grown in monolayer culture and tested for the presence of steroid hormone receptors and sensitivity to various steroids and steroid antagonists. None of the 10 solid tumors or the primary cultures derived from them contained estrogen receptors, either in the cytoplasm or in the nucleus. Progesterone receptors were present in 50-70% of the solid tumors and some of the primary cultures. Four of four and five of five primary cultures contained, respectively, androgen and glucocorticoid receptors. When one of the primary cultures was tested for growth sensitivity to estrogen, tamoxifen, progesterone, hydrocortisone, and dihydrotestosterone, the last two had noticeable stimulatory effects on growth by day 5. Interestingly, only androgen and glucocorticoid receptors were present in the primary tumor cells in culture, suggesting that these receptors mediated the effects of their respective hormones on growth.

Estrogen receptor in malignant melanoma: fact or artefact?

A model system is presented to explain how tyrosinase, an enzyme unique to pigmented cells such as normal and malignant melanocytes can oxidize [3H]-estradiol to radiolabeled products which closely resemble the tight binding of [3H]-estradiol to estrogen receptor. In the model system studied, tyrosinase oxidized [2,4,6,7-3H]-estradiol to [3H]-water and [3H]-estradiol metabolites, the latter of which formed ring-substituted conjugates with nucleophiles like monothioglycerol and BSA. Radiolabeled estradiol without tritium in the C-2 position (i.e. [6,7-3H]-estradiol) failed to liberate [3H]-water when exposed to tyrosinase but, nevertheless, did form ring-substituted [3H]-estradiol adducts with nucleophiles. The [3H]-water and the ring-substituted radiolabeled products possessed several characteristics of a genuine estrogen receptor protein in that they were resistant to dextran-coated charcoal (DCC) adsorption and their enzymatic formation was inhibited with non-steroidal estrogens like diethylstilbestrol. Other natural (estradiol) and synthetic (hydroxytamoxifen) estrogens which contain the phenol grouping also inhibited the enzymatic oxidation of [3H]-estradiol. Although it was difficult to differentiate estrogen receptor from tyrosinase using the conventional DCC assay system, several differences in these two proteins permitted a distinction to be made between them. First, tyrosinase oxidation of [3H]-estradiol was markedly inhibited by sulfhydryl reducing agents (monothioglycerol) that stabilize [3H]-estradiol binding to estrogen receptor. Second, estrogen receptor adsorbed by hydroxylapatite whereas tyrosinase did not, thus permitting the separation of these two proteins prior to incubation with [3H]-estradiol. We conclude that the [3H]-estradiol binding components in melanoma previously reported to be estrogen receptor probably represent instead the radiolabeled products of the tyrosinase-catalyzed oxidation of [3H]-estradiol.