Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.     

Estrogen receptor affinity and effects on MCF-7 cell growth of triarylethylene carboxylic acids related to tamoxifen.

The estrogen receptor binding, and growth suppressant and stimulating effects in MCF-7 human breast cancer cells, of four structural variants of the triarylethylene antiestrogen tamoxifen (1) were studied. In these analogs, the dialkylaminoethoxy side chain of 1 was replaced by carboxylic acid or oxyacetic acid substituents. The presence of a p-hydroxy group in the ring geminal to the one bearing the side chain resulted in ligands with estrogen receptor affinities greater than that of 1 but less than that of estradiol. Compared to 1, none of the test compounds were effective suppressants of cell growth. To the contrary, the phenolic oxyacetic acid analog effectively reversed the growth suppressive effect of 1. Also, it was as effective as estradiol, though less potent, in stimulating growth of cells grown in estrogen depleted medium, suggestive of full estrogen agonist activity. Its carboxylic acid counterpart had little or no effect on proliferation. Because the phenolic oxyacetic acid is a metabolite of 1 in animals, its estrogenicity may have therapeutic implications of concern, depending on the extent to which it is formed and distributed in tissues of patients receiving 1.     

Image cytometry of progesterone receptor expression during the cell cycle in the MCF-7 cell line

Progesterone receptors (PR) appear to be distributed in a heterogeneous way in mammary tumor cells. The study presented here was designed to examine if heterogeneity of PR expression is cell-cycle dependent. Immunofluorescence techniques were used to label PR on the MCF-7 human breast cancer cell line and image cytometry was used to analyze the PR expression during G0 (Ki-67 antigen-negative cells), G1, S, and G2/M cell-cycle phases. A second PR, BrdU, and DNA analysis was performed to study PR expression in the S-phase (BrdU-positive cells). Our results show that PR synthesis occurs preferentially during the G0-G1 transition and that PR levels are constant during the G1-G2 transition. The PR expression appears to be cell-cycle related and may therefore explain the heterogeneity of PR expression. However, the possibility that PR heterogeneity may be linked to the existence of PR-negative subclones cannot be ruled out.     

Estrogen and progesterone effects on transcapillary fluid dynamics

The purpose of this study was to determine estrogen (E(2)) and progesterone (P(4)) effects on atrial natriuretic peptide (ANP) control of plasma volume (PV) and transcapillary fluid dynamics. To this end, we suppressed reproductive function in 12 women (age 21-35 yr) using a gonadotropin releasing-hormone (GnRH) analog (leuprolide acetate) for 5 wk. During the 5th week, the women either received 4 days of E(2) administration (17beta-estradiol, transdermal patch, 0.1 mg/day) or 4 days of E(2) with P(4) administration (vaginal gel, 90 mg P(4) twice per day). At the end of the 4th and 5th week of GnRH analog and hormone administration, we determined PV (Evans blue dye) and changes in PV and forearm capillary filtration coefficient (CFC) during a 120-min infusion of ANP (5 ng x kg body wt(-1) x min(-1)). Preinfusion PV was estimated from Evans blue dye measurement taken over the last 30 min of infusion based on changes in hematocrit. E(2) treatment did not affect preinfusion PV relative to GnRH analog alone (45.3 +/- 3.1 vs. 45.4 +/- 3.1 ml/kg). During ANP infusion CFC was greater during E(2) treatment compared with GnRH analog alone (6.5 +/- 1.4 vs. 4.9 +/- 1.4 microl. 100 g(-1) x min(-1) mmHg(-1), P < 0.05). The %PV loss during ANP infusion was similar for E(2) and GnRH analog-alone treatments (-0.8 +/- 0.2 and -1.0 +/- 0.2 ml/kg, respectively), indicating the change in CFC had little systemic effect on ANP-related changes in PV. Estimated baseline PV was reduced by E(2)-P(4) treatment. During ANP infusion CFC was approximately 30% lower during E(2)-P(4) (6.0 +/- 0.5 vs. 4.3 +/- 4.3 microl. 100 g(-1) x min(-1) mm Hg(-1), P < 0.05), and the PV loss during ANP infusion was attenuated (-0.9 +/- 0.2 and -0.2 +/- 0.2 ml/kg for GnRH analog-alone and E(2)-P(4) treatments, respectively). Thus the E(2)-P(4) treatment lowered CFC and reduced PV loss during ANP infusion.     

Studies on type II progesterone receptor in MCF-7 cells

These experiments demonstrate for the first time the existence of a Type II progesterone receptor (RpII) in MCF-7 human breast cancer cells. RpII was shown to have a lower affinity for tritiated progesterone ([3H]Pg) (Kd greater than or equal to 13 nM) than classical Rp (Kd less than or equal to 3 nM). RpII was detected by cytosolic, nuclear, and whole cell assays of MCF-7 cells. Scatchard analysis of [3H]Pg binding data revealed that classical Rp but not RpII could be recompartmentalized from the cytosolic to the nuclear pool by treating cells 1 h at 37 degrees C with 1 microM Pg. RpII levels were shown to be increased more than two-fold by growing MCF-7 cells for 4 days in 10 nM estradiol (E2) plus 100 nM Pg when compared to either untreated cells or to cells treated with only E2.     

Chemical constituents from Neopestalotiopsis clavispora culture medium with estrogenic effects in MCF-7 cells

Estrogen is a vital hormone responsible for the development of the female reproductive system. Hence, estrogen deficiency in menopause increases various symptoms. This study aimed to discover biologically active compounds from an EtOAc-extract of Neopestalotiopsis clavispora culture medium which has previously shown estrogenic activity. A new α-pyrone (1) and a new tetramic acid derivative (2), together with hymenosetin (3) and pestalotiollide B (4), were isolated from the extract. The chemical structures of the new compounds were elucidated using MS and NMR spectroscopy, and the absolute configurations were established by quantum mechanical calculations of electronic circular dichroism and gauge-including atomic orbital NMR chemical shift, followed by DP4 + analysis. The isolated compounds were initially tested for their estrogenic activities using MCF-7 estrogen responsive human breast cancer cells. Hymenosetin (3) showed estrogenic activity by increasing the proliferation of MCF-7 cells at the concentration of 2.5 μM via the phosphorylation of estrogen receptor-α.     

Bisphenol A prevents MCF-7 breast cell apoptosis via the inhibition of progesterone receptor transactivation

2,2-Bis(4-hydroxyphenyl)propane (bisphenol A; BPA) is an environmental endocrine-disrupting chemical. It mimics the effects of estrogen at multiple levels by activating estrogen receptors (ERs); however, BPA also affects the proliferation of human breast cancer cells independent of ERs. Although BPA inhibits progesterone (P4) signaling, the toxicological significance of its effects remain unknown. Tripartite motif-containing 22 (TRIM22) has been identified as a P4-responsive and apoptosis-related gene. Nevertheless, it has not yet been established whether exogenous chemicals change TRIM22 gene levels. Therefore, the present study investigated the effects of BPA on P4 signaling and TRIM22 and TP53 expression in human breast carcinoma MCF-7 cells. In MCF-7 cells incubated with various concentrations of P4, TRIM22 messenger RNA (mRNA) levels increased in a dose-dependent manner. P4 induced apoptosis and decreased viability in MCF-7 cells. The knockdown of TRIM22 abolished P4-induced decreases in cell viability and P4-induced apoptosis. P4 increased TP53 mRNA expression and p53 knockdown decrease the basal level of TRIM22 and P4 increased TRIM22 mRNA expression independent of p53 expression. BPA attenuated P4-induced increases in the ratio of cell apoptosis in a concentration-dependent manner, and the P4-induced decreases in cell viability was abolished in the presence of 100 nM and higher BPA concentrations. Furthermore, BPA inhibited P4-induced TRIM22 and TP53 expression. In conclusion, BPA inhibited P4-induced apoptosis in MCF-7 cells via the inhibition of P4 receptor transactivation. TRIM22 gene has potential as a biomarker for investigating the disruption of P4 signaling by chemicals.     

Estrogen stimulation of progesterone synthesis by porcine granulosa cells in culture

Estrogen stimulates synthesis of progesterone by porcine granulosa cells in tissue culture. This enhancement is inhibited by cycloheximide, suggesting that protein synthesis is required for the effect. Estrogen is synergistic with hCG in increasing progesterone synthesis. Sequential treatment with hCG followed by estradiol causes maximal stimulation of progesterone production.     

Estrogen modifies the temperature effects of progesterone.

To test the hypothesis that progestin-mediated increases in resting core temperature and the core temperature threshold for sweating onset are counteracted by estrogen, we studied eight women (24 +/- 2 yr) at 27 degrees C rest, during 20 min of passive heating (35 degrees C), and during 40 min of exercise at 35 degrees C. Subjects were tested four times, during the early follicular and midluteal menstrual phases, after 4 wk of combined estradiol-norethindrone (progestin) oral contraceptive administration (OC E+P), and after 4 wk of progestin-only oral contraceptive administration (OC P). The order of the OC P and OC E+P were randomized. Baseline esophageal temperature (T(es)) at 27 degrees C was higher (P < 0.05) in the luteal phase (37.08 +/- 0.21 degrees C) and in OC P (37.60 +/- 0.31 degrees C) but not during OC E+P (37.04 +/- 0.23 degrees C) compared with the follicular phase (36.66 +/- 0.21 degrees C). T(es) remained above follicular phase levels throughout passive heating and exercise during OC P, whereas T(es) in the luteal phase was greater than in the follicular phase throughout exercise (P < 0.05). The T(es) threshold for sweating was also greater in the luteal phase (38.02 +/- 0.28 degrees C) and OC P (38.07 +/- 0.17 degrees C) compared with the follicular phase (37.32 +/- 0.11 degrees C) and OC E+P (37.46 +/- 0.18 degrees C). Progestin administration raised the T(es) threshold for sweating during OC P, but this effect was not present when estrogen was administered with progestin, suggesting that estrogen modifies progestin-related changes in temperature regulation. These data are also consistent with previous findings that estrogen lowers the thermoregulatory operating point.     

Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation

Background  

It has been previously suggested that angiogenesis occurs during the menstrual cycle. Moreover, a rise in uterine blood flow is largely maintained by vasodilatation and substantial increases in angiogenesis. It is known that estradiol (E2) and progesterone (P4) are involved in angiogenesis. Recently, endothelial progenitor cells (EPCs) were found to be involved in neovascularization; however, their roles in uterine neovascularization have not been well characterized. We hypothesized that E2- or P4-mediated EPC proliferation plays important roles in uterine neovascularization during the menstrual cycle.     

The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR.

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.     

Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells

The role of estrogen in the motility and invasion of breast cancer cells is controversial. Although estrogen receptor (ER)-positive breast tumors are considered less aggressive and more differentiated they still undergo metastasis. In many types of epithelial cancers, the ability to undergo metastasis has been associated with a loss of epithelial features and acquisition of mesenchymal properties leading to migration of individual cells, a process known as epithelial-to-mesenchymal transition (EMT). In this report, we show that a subset of ER-positive breast cancer cells can acquire mesenchymal-like features and motility in a reversible manner. In MCF-7 breast cancer cells estrogen-promoted acquisition of mesenchymal-like features while antiestrogens, such as tamoxifen, prevented this transition. Moreover, pharmacological inhibition of Src family kinases decreased the ability of estrogen to promote epithelial-to-mesenchymal-like transition. In addition to mesenchymal-like motility, a subset of estrogen-treated cells also moved as cell clusters (collective motility). While membrane localization of E-cadherin/beta-catenin was decreased in fibroblast-like cells, enhanced levels of E-cadherin/beta-catenin were detected in motile cell clusters. Thus, during tumor progression, estrogen may foster motility and invasion of ER-positive breast cancer by promoting simultaneously reversible EMT-like changes and collective motility. These studies suggest that antiestrogen therapy and Src family kinase inhibitors may decrease development of metastases in ER-positive breast cancer by blocking estrogen-dependent migration of human breast cancer cells.     

Cis-trimethoxystilbene,exhibits higher genotoxic and antiproliferative effects than its isomer trans-trimethoxystilbene in MCF-7 and MCF-10A cell lines

Stilbenes are a class of natural compounds with a wide variety of biological effects, such as antitumor activity. The best-known stilbene is resveratrol, whose clinical application is limited due to its low bioavailability. Methoxylated derivatives of this stilbene, including cis-trimethoxystilbene (cis-TMS) and trans-trimethoxystilbene (trans-TMS) have demonstrated more pronounced cytotoxic and anti-proliferative effects than resveratrol. Thus, the objective of this study is to evaluate and compare the cytotoxicity and antiproliferative effects of cis- and trans-TMS in MCF-7 and its normal counterpart MCF-10A. Both compounds were cytotoxic, genotoxic, and induced G2-M accumulation and cell death in the two cell lines. These results suggested that the genotoxicity of cis- and trans-TMS is involved in the reduction of cellular proliferation of MCF-7 and MCF-10A cells, but notably, such antiproliferative effects are more pronounced for cis- than trans-TMS.     

Estrogen and progesterone receptors in gynecomastia

The etiology of gynecomastia is unknown. There seems to be no increased incidence of malignancies in patients with idiopathic gynecomastia; however, patients with Klinefelter syndrome exhibit an increased incidence of malignancy. The authors reviewed the results of 34 patients with gynecomastia diagnosed in adolescence who, following initial evaluation, had a mastectomy. The estrogen and progesterone receptors were analyzed in these patients. Three of the patients were diagnosed with Klinefelter syndrome. These three patients exhibited elevated amounts of estrogen and progesterone receptors. None of the patients who were not diagnosed with this syndrome demonstrated significant elevation of their estrogen or progesterone receptors. The presence of elevated estrogen and progesterone receptors in patients with Klinefelter syndrome provides a potential mechanism by which these patients may develop breast neoplasms. The absence of elevated estrogen and progesterone receptors in patients with idiopathic gynecomastia may serve to clarify why these patients' disease rarely degenerates into malignancy.     

Additive growth-inhibitory effects of DL-alpha-difluoromethylornithine and antiestrogens on MCF-7 breast cancer cell line

We studied the growth inhibitory effects of DL-alpha-difluoromethylornithine, and antiestrogens (tamoxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, and LY117018) as single agents and in combinations on the proliferation of a breast cancer cell line, MCF-7. At 0.1 mM difluoromethylornithine, the proliferation of MCF-7 cells was inhibited to 75 +/- 6% of the controls. Treatment of the cells with 0.1 microM 4-hydroxytamoxifen reduced cell growth to 72 +/- 4%. Combination of 0.1 mM difluoromethylornithine and 0.1 microM 4-hydroxytamoxifen reduced cell growth to 38 +/- 5%, indicating additive growth inhibitory effects. Similar additive effects were observed with all 5 antiestrogens in combination with difluoromethylornithine.     

Interaction of Vault Particles with Estrogen Receptor in the MCF-7 Breast Cancer Cell

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.     

Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas

The control of immune responses by sex hormones is well documented but the effect of sex hormones on lymphoid cell subsets is poorly understood. We have investigated the expression of receptors for androgens (AR), estradiol (ER) and progesterone (PR) by human cell lines of the B lymphocyte lineage and by murine myeloma or hybridomas. AR, ER and PR were determined by cytosol and nuclear binding assays. Eleven human lymphoblastoid cell lines obtained by in vitro infection of blood or tonsil B cells with Epstein-Barr Virus (EBV) B95, did not express AR or ER. Similarly, 10 Burkitt's lymphoma cell lines were AR, ER and PR negative with the exception of the pre-B RAJI cells which bear AR. Among 13 cell lines derived from patients with multiple myeloma none expressed AR but five were found to bear ER (20-164 fmol/mg DNA or 5-10 fmol/mg protein). Four of the latter group also bear PR (86-450 fmol/mg DNA). Two mouse hybridomas out of seven tested were ER and PR positive. The MOPC 315 myeloma expressed ER but not PR. The possible functional role of these sex hormone binding sites in cell proliferation and immunoglobulin secretion deserves further investigation.     

DNA interactive property of poly-L-lysine induces apoptosis in MCF-7 cells through DNA interaction

Poly-L-lysine (PLL) is known to be an encapsulating agent in drug formulation and delivery. PLL also has apoptotic and antiproliferative activities that enable blocking of the tumorigenesis process. However, the dose-selective activities of PLL in exerting apoptosis against cancer are unclear. Therefore, this study has been designed to explore the potential role and dose of PLL in apoptosis, if any. For this, PLL was administered at several doses in cancer cell lines and was found to be more potent against MCF-7 cells. PLL causes mitochondria-mediated apoptotic death through the upregulation of cleaved caspase-3. To investigate the mechanism responsible for this activity, we have analyzed if PLL could have the DNA interactive property or not. For this, molecular docking analysis was carried out to prove whether it has the property to bind with DNA or not. Studies have revealed that PLL is a potent DNA binder and it probably performs such apoptotic activities through the binding of cellular DNA early in an exposure. Simultaneous upregulation of both ROS-mediated stress and also in key protein expressions like γ-H2AX could also help us to confirm that PLL induces apoptosis through DNA interaction. This finding leads us to believe that PLL could play an interfering role with other chemotherapeutic compounds when used as a drug-coating material as it exerts an apoptotic effect on cancer cells, which should be avoided by using a much lower concentration.     

In vitro effects of 2-methoxyestradiol on MCF-12A and MCF-7 cell growth, morphology and mitotic spindle formation

The influence of 2-methoxyestradiol (2ME) was investigated on cell growth, morphology and spindle formation in a tumorigenic (MCF-7) and non-tumorigenic (MCF-12A) epithelial breast cell line. Inhibition of cell growth was more pronounced in the MCF-7 cells compared to the MCF-12A cells following 2ME treatment. Dose-dependent studies (10(-5)-10(-9) M) revealed that 10(-6) M 2ME inhibited cell growth by 44% in MCF-12A cells and by 84% in MCF-7 cells (p-value < 0.05). 2ME-treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent or less prominent in MCF-12A cells. 2ME had no effect on the length of the cell cycle between S-phase and the time a mitotic peak was reached in either cell line but MCF-7 cells were blocked in mitosis with no statistically significant alterations in the phosphorylation status of Cdc25C. Nevertheless, Cdc2 activity was significantly increased in MCF-7 cells compared to MCF-12A cells (p-value < 0.05). The results indicate that 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest that may result in the induction of apoptosis. The tumorigenic MCF-7 cells were especially sensitive to 2ME treatment compared to the normal MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells.