Estrogen regulation of methyl p-hydroxyphenyllactate hydrolysis: correlation with estrogen stimulation of rat uterine growth

We have recently demonstrated that methyl p-hydroxyphenyllactate (MeHPLA) is the endogenous ligand for nuclear type II binding sites in the rat uterus and other estrogen target and non-target tissues. MeHPLA binds to nuclear type II binding sites with a very high binding affinity (Kd approximately 4-5 nM), blocks uterine growth in vivo, and inhibits MCF-7 human breast cancer cell growth in vitro. Conversely, the free acid (p-hydroxyphenyllactic acid, HPLA) interacts with type II binding sites with a much lower affinity (Kd approximately 200 nM) and does not inhibit estrogen-induced uterine growth in vivo or MCF-7 cell growth in vitro. On the basis of these observations, we suggested that one way that estrogen may override MeHPLA inhibition of rat uterine growth may be to stimulate esterase hydrolysis of MeHPLA to HPLA. The present studies demonstrate that the rat uterus does contain an esterase (mol. wt approximately 50,000) which cleaves MeHPLA to HPLA, and that this enzyme is under estrogen regulation. This conclusion is supported by the observations that MeHPLA esterase activity is increased 2-3-fold above controls within 2-4 h following a single injection of estradiol, and is maintained at high levels for 16-24 h following hormone administration. This sustained elevation of MeHPLA esterase activity correlates with estradiol stimulation of true uterine growth and DNA synthesis.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Nuclear type II [3H]estradiol binding site ligands: inhibition of ER-positive and ER-negative cell proliferation and c-Myc and cyclin D1 gene expression

These studies assessed the effects of 3,4-dihydroxybenzalacetone (ZN-1) and 1-(3,4-dihydroxyphenyl)-2-propanol (ZN-2) on MCF-7 cell proliferation. The compounds blocked [3H]estradiol binding to nuclear type II sites, but did not compete for [3H]estradiol binding to recombinant ERalpha or ERbeta. ZN-1 and ZN-2 inhibited the proliferation of ERalpha and ERbeta positive (MCF-7) and negative (MCF-10A) breast cells, further ruling out direct binding to ER in the mechanism of action of these compounds. Pre-loading type II sites with ZN-1 or ZN-2 reduced [3H]estradiol exchange, strongly suggesting the drugs were binding covalently. ZN-1 treatment resulted in complete occupancy of type II sites and sustained (9 days) inhibition of MCF-7 cell proliferation following its removal from the tissue culture medium. This cell growth inhibition was not due to non-specific toxicity, as the numbers of viable, attached cells per dish (determined by trypan blue dye exclusion) remained constant throughout this 9-day period and eventually reversed by day 19. ZN-2 effects on cell proliferation reversed more rapidly following discontinuation of treatment, a response consistent with the inability of the compound to totally block type II binding. Both ZN-1 and ZN-2 blocked estradiol stimulation of c-Myc and cyclin D1 gene expression in MCF-7 cells, two events that are clearly coupled to cell cycle progression. We suspect this may occur through ZN-1 or ZN-2 modification of nucleosome function and/or chromatin remodeling since nuclear type II sites are localized to a complex of histones H3 and H4 (Shoulars et. al, J Steroid Biochem. Mol. Biol. 96: 19-30, 2005).     

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.     

Type II estrogen binding site agonist: synthesis and biological evaluation of the enantiomers of methyl-para-hydroxyphenyllactate (MeHPLA)

A high-affinity ligand for the type II estrogen binding site (EBS) was identified. Methyl para-hydroxyphenyllactate (MeHPLA) was observed to suppress the cellular proliferation of estrogen-sensitive MCF-7 breast cancer cells in vitro and to suppress the growth of rat uteri in vivo. The high affinity of MeHPLA for the type II EBS suggests that this interaction is responsible for the observed suppression of cell growth. In this study, the enantiomers of MeHPLA were synthesized and separated by three methods and evaluated for biological activity. When the methods were compared, it was found that the method using an optically pure amine to form the diastereomers of the enantiomers gave the superior yield. Binding studies for the enantiomers to the type II EBS showed that the S-MeHPLA had a higher affinity for the binding site. However, higher binding affinity did not translate into superior cell growth suppression. Both enantiomers suppressed cell growth equally.     

Cytosol type II sites in the rat uterus: interaction with an endogenous ligand

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.     

Reconstitution of the type II [3H]estradiol binding site with recombinant histone H4

Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd approximately 20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient approximately 4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.     

Effects of Salt Extraction on the Quantitation of Nuclear Estrogen Receptors: Interference by Secondary Estrogen Binding Sites

Abstract

The effects of salt-extraction on type I and type II estrogen binding sites were examined in uterine nuclei. Injection (10 ug) of estradiol or estriol in adult ovariectomized rats induced maximum numbers (80–100%, ~ 1 pmole/uterus) of 0.4 M KCL resistant type I estrogen complexes at 1 hour. Only estradiol, which sustained these levels for long periods of time (4–24 hours) stimulated true uterine growth.

Likewise, a single injection of estradiol, but not estriol, also elevated nuclear type II sites throughout the entire uterine growth period (1–48 hours). However extraction of these nuclei from estradiol injected rats with 0.4 M KCL increased the numbers of type II sites from ~ 1 pmole/uterus (non-extracted nuclei) to ~ 8 pmoles/uterus (salt resistant plus salt-extractable fractions). Sixty percent of these sites were resistant to salt-extraction. Continuous exposure to either estradiol or estriol by beeswax implants stimulated nuclear type II sites which were highly resistant (80%) to KCL-extraction, and additional sites were not exposed by high salt. Thus chronic treatment with both estrogens “locked in” nuclear type II sites such that they were resistant to KCL-extraction. This resistance of type II sites to salt-extraction correlated with the ability of estradiol and estriol implants to stimulate true uterine growth. The procedures presented here for nuclear preparation and assay have reduced non-specific binding considerably in the uterine system, and may eliminate the need to perform exchange assays on salt-extracted nuclei in other systems.     

Endocrine characteristics of human breast epithelial cells, MCF-10F

MCF-10F is a spontaneously immortalized nontransformed human breast epithelial cell line which does not grow in soft agar or form tumors in nude mice. Though the presence of estrogen receptors has not been found in these cells, they can metabolize estradiol very efficiently. The present study describes the endocrine characteristics of this cell line with respect to growth response to estradiol and its metabolites, estradiol metabolism and aromatase activity. MCF-10F cells were growth stimulated by 16alpha-hydroxyestrone and estriol, whereas, estradiol and other estradiol metabolites did not affect cell proliferation. The constitutive level of 16alpha-hydroxyestrone, a metabolite of estradiol biotransformation that has been associated with enhanced carcinogenesis in several animal, cell and tissue culture models, was a hundredfold higher in the non-transformed MCF-10F cells than in the transformed MCF-7 cells. Treatment with the carcinogen, dimethylbenz(a)anthracene (DMBA), however, did not upregulate 16alpha-hydroxylation as was observed in transformed MCF-7 cells. MCF-10F cells also had no detectable aromatase activity though the level of 17-oxidation was unusually high as compared with MCF-7 cells. Our results using the non-transformed MCF-10F cells as a model system suggests that the presence of high level of 16alpha-hydroxyestrone, a metabolite previously shown to be associated with malignant phenotype, may not be sufficient for breast cancer transformation.     

Plasma membrane receptors for the cancer-regulating progesterone metabolites, 5alpha-pregnane-3,20-dione and 3alpha-hydroxy-4-pregnen-20-one in MCF-7 breast cancer cells

Recent observations indicate that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), which is produced at higher levels in tumorous breast tissue, promotes cell proliferation and detachment, whereas 3alpha-hydroxy-4-pregnen-20-one (3alphaHP), which is produced at higher levels in nontumorous breast tissue, suppresses proliferation and detachment of MCF-7 breast cancer cells. The objective of the current study was to determine the presence and characteristics of binding sites for these endogenous putative cancer-regulating steroid hormones. Radiolabeled 5alphaP and 3alphaHP were used in radioligand binding assays on MCF-7 cell (membrane, cytosolic, and nuclear) fractions. Binding of [(3)H]5alphaP and [(3)H]3alphaHP was observed only in the plasma membrane fraction, whereas estradiol binding sites were confirmed in the cytosolic and nuclear fractions. The respective membrane binding sites exhibited specificity for the 5alphaP and 3alphaHP ligands with no appreciable displacement at 200- to 500-fold excess by other steroids. The association rate constants were calculated as 0. 107/min and 0.0089/min and the dissociation rate constants were 0. 049 9 and 0.011 for 5alphaP and 3alphaHP, respectively. Saturation analyses indicated single classes of molecules with dissociation constants of 4.5 and 4.87 nM and receptor densities of 486 and 629 fmol/mg protein, respectively, for 5alphaP and 3alphaHP. Exposure of MCF-7 cells to estradiol for 1, 24, 48, and 72 h resulted in 2.3, 4. 2-, 2.99-, and 1.7-fold increases, respectively, in 5alphaP receptor density. 3alphaHP resulted in partial suppression of the estradiol-mediated increase in 5alphaP receptor density. This is the first report of receptors for the progesterone metabolites, 5alphaP and 3alphaHP, of their occurrence in breast cancer cell membranes, and of the induction of 5alphaP receptors by estradiol. The results provide further support for the potential importance of progesterone metabolites in breast cancer.     

Uterine type II estrogen-binding sites are not of eosinophil origin

A recent report by Lyttle et al. (Lyttle, C. R., Medlock, R. L., and Sheehan, D. M. (1984) J. Biol. Chem. 259, 2697-2700) suggested that nuclear type II sites in the rat uterus are of eosinophil origin and may represent [3H]estradiol binding to eosinophil peroxidase. To further evaluate this hypothesis we examined the response of nuclear type II sites to estrogen under conditions where eosinophils are not present. Results of our experiments show that physiological levels of estradiol-17 beta (10 nM for 72 h) will stimulate nuclear type II sites in highly purified cultures (21-25 days; 4 passages) of rat uterine stromal and myometrial cells. The magnitude of the response of type II sites to estradiol in these stromal (4-fold) and myometrial (80-fold) cell cultures was essentially identical to that observed in the uterine cell types following in vivo estrogen treatment. Since these highly purified cultures of uterine cells were prepared from the uterus of a 21-day ovariectomized rat which is devoid of eosinophils, we conclude that estradiol stimulation of nuclear type II sites is a direct intracellular response to estrogen which occurs independent of eosinophil accumulation. Furthermore, we have found that type II sites in the rat uterus are not peroxidase. This was demonstrated by experiments which show type II sites are present in the 39,000 X g supernatant fraction of uterine cytosol, whereas peroxidase activity is quantitatively recovered in the crude mitochondrial (39,000 X g) pellet. Likewise, the small amount of peroxidase activity (approximately 10%) in the total homogenate which contaminates our nuclear pellet preparations was extracted (98-100%) with 0.5 M CaCl2. Type II estrogen-binding sites (95-100%) remained associated with the nuclear pellet fraction after peroxidase extraction. Therefore, stimulation of cytosol and nuclear type II sites by estrogen in the rat uterus is a direct intracellular response to the hormone unrelated to eosinophil accumulation and/or peroxidase activity.     

Platinum(II) complexes with steroidal esters of L-methionine and L-histidine: synthesis, characterization and cytotoxic activity

Twelve steroidal platinum(II) complexes were synthesized by reaction of potassium tetrachloroplatinate with steroidal esters of L-methionine and L-histidine. The steroidal esters coordinated as bidentate ligands via S and N donor atoms of L-methionine and via two N donor atoms of L-histidine. Cholesterol, cholestanol, diosgenine, pregnenolone, dehydroepiandrosterone, testosterone, estrone, and estradiol were used as the steroidal compounds. The esters and complexes prepared were characterized by infrared, mass, and (1)H NMR spectroscopy and elemental analysis. Platinum complexes were tested for in vitro cytotoxicity against several cancer cell lines: T-lymphoblastic leukemia CEM, breast carcinoma MCF-7, lung carcinoma A-549, multiple myeloma RPMI 8226, and one normal cell line human fibroblast BJ.     

Synthesis of chalcone derivatives on steroidal framework and their anticancer activities

Chalcone derivatives on estradiol framework have been synthesized. Some of the derivatives showed potent anticancer activity against some human cancer cell lines. Compounds 9 and 19 showed potent activity against MCF-7, a hormone dependent breast cancer cell line. Chalcone 7 was further modified to the corresponding indanone derivative (19) using the Nazarov reaction, which showed better activity than the parent compound against the MCF-7 breast cancer cell line. Active anticancer derivatives were also evaluated for osmotic hemolysis using the erythrocyte as a model system. It was observed that chalcone derivatives showing cytotoxicity against cancer cell lines did not affect the fragility of erythrocytes and hence may be considered as non-toxic to normal cells.     

Aromatase expression and its localization in human breast cancer

Aromatization or in situ estrogen production by aromatase has been considered to play an important role in the development of human breast carcinoma. In the human breast, aromatase overexpression is observed in the stromal or interstitial cells of the carcinoma, especially at the sites of frank invasion and/or adipose tissue. Transplantation experiments in the nude mouse employing MCF-7 and/or SF-TY human fibroblast cell lines revealed that aromatase activity and expression were much higher in the tumour with MCF-7 and SF-TY than that with MCF-7 alone. Aromatase overexpression in human breast carcinoma tissue is considered to occur as a result of carcinomastromal cell interactions, i.e. paracrine communication between stromal and carcinoma cells. Aromatase overexpression is correlated with the malignant phenotype in the human breast, but not with stage, age, clinical stages, clinical course, or proliferative activity of breast carcinoma. Aromatase overexpression may be correlated with development, rather than the biological behaviour of breast malignancy. Aromatase overexpression is not necessarily correlated with expression of 17β-hydroxysteroid dehydrogenase type 1, which converts estrone to estradiol and estrogen receptor. Different mechanisms may be involved in the regulation of expression of these two important estrogen-metabolizing enzymes and estrogen receptor in human breast cancer. Aromatase overexpression in intratumoral stromal cells was much more frequently detected in male breast cancer than in female counterparts, which confers a growth advantage on cancer cells in a male hormonal environment with low serum estrogen levels.     

Dose-dependent changes of the ratio of apoptosis to proliferation by norethisterone and medroxyprogesterone acetate in human breast epithelial cells.

OBJECTIVE: There is increasing evidence that adding progestogens to estrogen replacement therapy may do more harm than good; however, whether all progestogens act equally on breast cells is debatable. Apart from estrogens, mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify the response to progestogens. We investigated the effects of medroxyprogesterone acetate (MPA) as well as norethisterone (NET) in the presence of a growth factor mixture and/or estradiol in normal and cancerous human epithelial breast cells. METHODS: MCF10A cells (human epithelial, estrogen- and progesterone-receptor negative, normal breast cells), HCC1500 (human estrogen and progesterone receptor-positive primary breast cancer cells) and MCF-7 cells (human estrogen and progesterone receptor-positive metastatic breast cancer cell line) were used in the experiments. The cells were incubated with progestogens at concentrations of 10(-10) to 10(-6) M for 7 days and growth factors (GFs), estradiol (E2) alone and a combination of GFs + E2. Cell proliferation rate was measured by ATP assay. Apoptosis was measured by cell death assay. Ratios of cell death : proliferation were calculated from these results. RESULTS: In MCF10A cells growth factors elicited a decrease in the ratio of apoptosis to proliferation. This effect was further stimulated by the addition of MPA, whereas NET had no effect. In HCC cells growth factors and estradiol alone and in combination led to a reduction in the ratio. This effect could be partly reversed dose-dependently by the addition of MPA and NET, being more pronounced for MPA. Similar results were found for MCF-7 cells stimulated by estradiol. CONCLUSION: The results of our investigations demonstrate that there are differences between the two progestogens NET and MPA investigated with respect to their effects on normal and cancerous cells. By increasing the mitotic rate of normal epithelial cells, MPA may increase breast cancer risk in women when used in long-term treatment. In this respect NET reacts neutral. The mitosis of pre-existing cancerous cells may be partly inhibited by the addition of both progestogens. Thus, our results indicate that it is necessary to differentiate between normal and malignant breast cells concerning the assessment of progestogens as a risk factor for breast carcinogenesis.     

Abrogation of TGFbeta signaling induces apoptosis through the modulation of MAP kinase pathways in breast cancer cells

Transforming growth factor beta (TGFbeta) can modulate the activity of various MAP kinases. However, how this pathway may mediate TGFbeta-induced malignant phenotypes remains elusive. We investigated the role of autocrine TGFbeta signaling through MAP kinases in the regulation of cell survival in breast carcinoma MCF-7 cells and untransformed human mammary epithelial cells (HMECs). Our results show that abrogation of autocrine TGFbeta signaling with the expression of a dominant negative type II TGFbeta receptor (DNRII) or the treatment with a TGFbeta type I receptor inhibitor significantly increased apoptosis in MCF-7 cell, but not in HMEC. The expression of DNRII markedly decreased activated/phosphorylated Erk, whereas increased activated/phosphorylated p38 in MCF-7 cells. In contrast, there was no or little change of phosphorylated Erk and p38 in HMECs after the expression of DNRII. Inhibition of Erk activity in MCF-7 control cell induced apoptosis whereas restoration of Erk activity in MCF-7 DNRII cell reduced apoptosis. Similarly, inhibition of p38 activity also inhibited apoptosis in MCF-7 DNRII cell. Thus, autocrine TGFbeta signaling can enhance the survival of MCF-7 cells by maintaining the level of active Erk high and the level of active p38 low. Furthermore, the survival properties of TGFbeta pathway appear related to transformation supporting the notion that it may be a potential target for cancer therapy.