Anti-proliferative and anti-estrogenic effects of ICI 164,384 and ICI 182,780 in 4-OH-tamoxifen-resistant human breast-cancer cells

The effects of the anti-estrogens 4-hydroxytamoxifen (OHTam), ICI 164,384 and ICI 182,780 were tested on the MCF-7/LCC2 breast-carcinoma cell line, which grows significantly in the presence of OHTam and serves as a model for studying anti-estrogen resistance of estrogen-receptor-positive breast cancer. Cell proliferation and cathepsin-D secretion were strongly inhibited by either ICI 182,780 or ICI 164,384 alone or ICI 164,384 in combination with 17-p-estradiol (E2) or OHTam. ICI 164,384 alone did not affect the cathepsin-D and pS2 mRNA levels, but antagonized the stimulatory effects of E2 or OHTam on these 2 mRNAs. OHTam was more effective than E2 in increasing cathepsin-D mRNA levels, supporting the idea that anti-estrogen-resistant breast cancer continues to over-express cathepsin-D. These data show that the steroidal anti-estrogens ICI 164,384 and ICI 182,780 retain their ability to inhibit cell proliferation and the estrogen-responsiveness of cathepsin-D and pS2 genes in the OHTam-resistant MCF-7/ LCC2 cell line. These pure anti-estrogens may thus be efficient second-line treatments of some Tamoxifen-resistant tumors.     

A potent specific pure antiestrogen with clinical potential.

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells. Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.     

Circumvention of tamoxifen resistance by the pure anti-estrogen ICI 182, 780

Both primary and acquired resistance to the growth-inhibitory effects of anti-estrogens (e.g., tamoxifen) limits the clinical usefulness of these drugs in the treatment of breast cancer. The new, steroidal anti-estrogen ICI 182,780 was tested for its ability to inhibit the proliferation of a tamoxifen-resistant variant of the parental MCF-7 human breast-cancer cell line. Two cell lines cloned from the MCF-7 line were used for these experiments: a tamoxifen-sensitive line, MCF 5-21, and a tamoxifen-resistant line, MCF 5-23. Compared with tamoxifen, ICI 182,780 appeared to be 150 and 1540 times more effective in inhibiting cell growth in the 5-21 and 5-23 sub-lines respectively. ICI 182,780 completely circumvented tamoxifen resistance at a concentration of (5 to 10) × 10^?9 M in this model. Based on IC₅₀ concentrations, the 5-23 line was 22-fold more resistant to tamoxifen than the 5-21 line, but only 2-fold more resistant to ICI 182, 780, reducing relative resistance by 10-fold in the resistant line. There were no differences in ER parameters between the 2 lines. ER numbers/cell were: 40500 and 34800 and the K_D 0.48 and 0.15 × 10^?9 M in the 5-21 and 5-23 cells respectively. In the 5-23 cells, the concentrations of ICI 182, 780 and tamoxifen resulting in a 50% inhibition of ³ H-estradiol binding were 2.3 × 10^?8 M and 1 × 10^?6 M, respectively (cf. estradiol 0.89 × 10^?9 M). Thus, one potential mechanism for the increased effectiveness of ICI 182,780 may relate to the increased affinity of this drug for the estrogen receptor as compared with tamoxifen.     

Altered radiation responses of breast cancer cells resistant to hormonal therapy

Endocrine therapy agents (the selective estrogen receptor (ER) modulators such as tamoxifen or the selective ER down-regulators such as ICI 182,780) are key treatment regimens for hormone receptor-positive breast cancers. While these drugs are very effective in controlling ER-positive breast cancer, many tumors that initially respond well to treatment often acquire drug resistance, which is a major clinical problem. In clinical practice, hormonal therapy agents are commonly used in combination or sequence with radiation therapy. Tamoxifen treatment and radiotherapy improve both local tumor control and patient survival. However, tamoxifen treatment may render cancer cells less responsive to radiation therapy.Only a handful of data exist on the effects of radiation on cells resistant to hormonal therapy agents. These scarce data show that cells that were resistant to tamoxifen were also resistant to radiation. Yet, the existence and mechanisms of cross-resistance to endocrine therapy and radiation therapy need to be established.Here, we for the first time examined and compared radiation responses of MCF-7 breast adenocarcinoma cells (MCF-7/S0.5) and two antiestrogen resistant cell lines derived from MCF-7/S0.5: the tamoxifen resistant MCF-7/TAM^R-1 and ICI 182,780 resistant MCF-7/182^R-6 cell lines. Specifically, we analyzed the radiation-induced changes in the expression of genes involved in DNA damage, apoptosis, and cell cycle regulation. We found that the tamoxifen-resistant cell line in contrast to the parental and ICI 182,780-resistant cell lines displayed a significantly less radiation-induced decrease in the expression of genes involved in DNA repair. Furthermore, we show that MCF-7/TAM^R-1 and MCF-7/182^R-6 cells were less susceptible to radiation-induced apoptosis as compared to the parental line. These data indicate that tamoxifen-resistant breast cancer cells have a reduced sensitivity to radiation treatment. The current study may therefore serve as a roadmap to the future analysis of the mechanisms of cross-resistance between hormonal therapy and radiation.     

Modulatory effect of tamoxifen and ICI 182,780 on adriamycin resistance in MCF-7 human breast-cancer cells

In this study the ability of the new pure anti-estrogen ICI 182,780 to modulate the cytotoxic action of adriamycin (ADR) on parental and ADR-resistant MCF-7 (MCF-7 ADRr) human breast-cancer cells was investigated and compared with that of tamoxifen (TAM). TAM enhanced ADR cytotoxicity in MCF-7 ADRr cells in a dose-related manner, but this effect was slight or absent in MCF-7 WT. In contrast, ICI 182,780 was able to enhance ADR toxicity both in MCF-7 ADRr and in the parental cell line. ICI 182,780 was up to 2.5-fold more effective than TAM in reducing the IC₅₀ of ADR in MCF-7 ADRr cells. Analysis of the data by the isobole method showed that the combination ADR/TAM and ADR/ICI 182,780 produced synergistic anti-proliferative activity in MCF-7 ADRr cells. Because ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of anti-estrogens on Pgp expression and activity. Both ICI 182,780 and TAM failed to modulate Pgp expression as assessed by flow cytometry and Western-blot analysis, performed using the monoclonal antibodies MM4.17 and C219, which are specific for an external or an internal determinant respectively. Pgp activity was investigated by flow cytometry measuring the extrusion of ADR and the cationic dye Rhodamine 123 (Rh 123). ICI 182,780, but not TAM, reduced the activity of Pgp in MCF-7 ADRr cells. Flow cytometry was also used to investigate cell-cycle modifications induced by ADR in MCF-7 ADRr cells, both in the presence and in the absence of anti-estrogens. After 72 hr, higher doses induced an arrest of cells at the G₂/M phase. The same effect was visible when lower doses of ADR were combined with ICI 182,780 or TAM. In terms of cell-cycle-blocking activity ICI 182,780 was largely more effective than TAM. © 1996 Wiley-Liss, Inc.     

Inhibition of growth-factor-activated proliferation by anti-estrogens and effects on early gene expression of mcf-7 cells

Recently, it was reported that the anti-estrogen tamoxifen not only inhibits estradiol-stimulated growth of MCF-7 cells but also significantly reduces the proliferation rate of cells stimulated by growth factors. We have confirmed this finding and also shown that the new anti-estrogen droloxifene inhibits the proliferation of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I)-stimulated MCF-7 cells. The growth-factor-induced proliferation was inhibited in a dose-dependent manner by the anti-estrogens in the complete absence of estrogen and FCS. Of the anti-estrogens, droloxifene was considerably more potent than tamoxifen. Because the exprersion of the proto-oncogenes c-fos and c-myc has been considered a key event in development of the mitogenic response, we examined the effects of anti-estrogens on c-myc and c-fos gene expression. We included in these investigations the steroidal anti-estrogen ICI 164,384 because this compound has no or very little estrogenic activity. The studies revealed that all 3 antiestrogens transiently induced c-myc mRNA expression. However, the anti-estrogens inhibited estradiol-induced c-myc mRNA expression, although with different potencies. Pre-incubation of MCF-7 cells with droloxifene and tamoxifen resulted in elevated levels of growth-factor-induced c-myc mRNA expression. In contrast, the anti-estrogens did not induce c-fos mRNA or affect the expression of c-fos mRNA induced by growth factors. In conclusion, non-steroidal anti-estrogens inhibit growth-factor-stimulated proliferation of MCF-7 cells without inhibitinggrowth-factor-induced c-myc or c-fos mRNA expression.     

Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family

Development of acquired resistance against antiestrogen treatment is a serious problem in human breast cancer, and knowledge of alterations resulting in resistance is important for selection of further treatment. To mimic the clinical situation we have established a series of MCF-7 human breast cancer cell lines by long term treatment with the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Common for these cell lines is a decreased expression of the estrogen receptor (ER). In human breast cancer, lack of response to endocrine therapy is often associated with decreased expression of the estrogen receptor and increased expression of epidermal growth factor receptor (EGFR) and/or HER-2/neu (ErbB-2). Our antiestrogen resistant cell lines did not express altered levels of EGFR, HER-2/neu, ErbB-3, or ErbB-4. Estrogen and antiestrogen regulation of HER-2/neu expression was essentially similar in parent and resistant MCF-7 cells. Treatment with antibodies to HER-2/neu (Herceptin^TM) did not affect growth of MCF-7 cells or resistant cells, indicating that in this in vitro model system, acquired antiestrogen resistance does not emerge from activation of the HER-2/neu signaling pathway. In MCF-7 cells transfected with HER-2/neu and/or ErbB-3, overexpression alone did not result in resistance. However, addition of heregulinl-1 abolished the inhibitory activity of ICI 182,780 on both vector and HER-2/neu/ErbB-3 transfected MCF-7 cells, demonstrating that activation of the HER-2/neu receptor signaling pathway can override the growth inhibitory effect of ICI 182,780.     

Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs. independent human breast cancer cell lines

Summary The effect of the anti-estrogens ICI 164,384 and tamoxifen on the estradiol (E₂) concentration after incubation of estrone sulfate (E₁-S) with different hormone-dependent (MCF-7 and T-47D) and hormone-independent (MDA-MD-231 and MDA-MB-436) mammary cancer cells, as well as the estrone sulfatase activity in these various cell lines, are presented. The anti-estrogen ICI 164,384 decreased very significantly the concentration of E₂ after incubation of E₁-S with MCF-7 (control, mean ±SE: 100±24 pg/mg DNA; + ICI 164,384 [10^–6M]: 7±2 pg/mg DNA). This effect was much more intense than with tamoxifen. A similar effect was observed with T-47D cells. However, no significant effect was observed in the hormone-independent cells. In the intact cell, estrone sulfatase activity was very intense in the hormone-dependent cells, but very small in the hormone-independent cells. However, this activity became very strong after homogenization in the hormone-independent cells. The data suggest that estrone sulfate can play an important role on the bioavailability of E₂ in hormone-dependent breast cancer, and that understanding the control of estrone sulfatase activity can open new knowledge of the estrogen responses and new possibilities of therapeutic application in breast cancer.     

Effect of estrogen and antiestrogens on cell proliferation and synthesis of secreted proteins in the human breast cancer cell line MCF-7 and a tamoxifen resistant variant subline, AL-1.

The human breast cancer cell line MCF-7 contains estrogen receptors and responds to estrogens with an increase in growth rate and to antiestrogens with a decrease in growth rate. Estrogen stimulation of cell proliferation is concomitant with an increase in the synthesis and secretion of three proteins with mol. wt 52 kDa, 61 kDa and 66 kDa and a decrease in the synthesis and secretion of a 42 kDa protein. The antiestrogen ICI 164,384 has a complete estrogen antagonistic effect on the synthesis of these secreted proteins, whereas the antiestrogen tamoxifen has an agonistic effect on the synthesis and secretion of the 52 kDa protein. We believe that the above mentioned estrogen regulated secreted proteins are either directly or indirectly involved in control of cell proliferation, and the less pronounced inhibitory effect of tamoxifen on cell proliferation compared to ICI 164,384 may be due to agonistic effects of tamoxifen. A tamoxifen resistant variant of the MCF-7 cell line, the AL-1 subline, can be growth inhibited by ICI 164,384, although a higher concentration is needed to inhibit the AL-1 cells compared to the parent MCF-7 cells. Tamoxifen has no effect on secreted proteins from the AL-1 cells, whereas ICI 164,384 has a complete estrogen antagonistic effect on secreted proteins, indicating that the mechanisms by which estrogens and antiestrogens influence cell proliferation may be via up and down regulation of secreted proteins with growth regulatory functions.     

Farnesyl-transferase inhibitor R115,777 enhances tamoxifen inhibition of MCF-7 cell growth through estrogen receptor dependent and independent pathways

Introduction

We have previously shown that FTI-277, a farnesyl transferase inhibitor (FTI), enhances the efficacy of tamoxifen (Tam) in inhibiting the proliferation of the estrogen dependent MCF-7 cell line. As the cellular response to Tam is the result of an inhibition of both estrogen receptor-dependent and -independent pathways, we have used the estrogen receptor selective anti-estrogen ICI182,780 and N-pyrrolidine(-phenylmethyl-phenoxy)-ethanamine-HCl (PBPE), a selective ligand of anti-estrogen binding site (AEBS), to dissect out the mechanism(s) associated with the observed additivity resulting from combination treatment with FTI-277 and Tam. Moreover, for these studies, FTI-277 has been replaced by R115,777, a FTI currently in phase III clinical trials.

Methods

The quantitative sulphorhodamine B (SRB) colorimetric assay was used to determine the growth inhibitory effect of agents on MCF-7 cells. Dose response interactions between R115,777-Tam, R115,777-ICI182,780 and R115,777-PBPE were evaluated, at the IC₅₀ point, using the isobologram method. Apoptotic cell death (DNA fragmentation, nucleus condensation and cytokeratin 18 cleavage) and inhibition of the mevalonate pathway (western blot) were also determined.

Results

Combinations of the specific FTI R115,777 with either ICI182,780 or PBPE exhibit a synergistic effect on MCF-7 cell growth inhibition, while its combination with Tam is additive, as previously reported for FTI-277. Apoptosis is detected after treatment with combinations of R115,777 with either Tam or PBPE but not with ICI182,780, suggesting that each combination inhibits cell proliferation by different mechanisms. Even though the ER pathway has not yet been deciphered, it is shown here that the AEBS pathway is able to interfere with the mevalonate pathway at the level of protein farnesylation.

Conclusion

Overall, this work reveals that combinations of R115,777 with either selective ER ligands or a selective AEBS ligand are able to induce large increases in their anti-proliferative activities on MCF-7 cells. Moreover, these results suggest that it may be of definite interest to evaluate combinations of R115,777 with different anti-estrogens in the treatment of ER positive breast tumours. Based on these experimental data, such combinations may prove beneficial in different clinical scenarios or when used in specific sequences; studying the combination of R115,777 with ICI182,780 for early treatment and reserving combinations with either Tam or a selective AEBS ligand, such as BMS-217380-01, for more resistant disease.     

Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo.

PURPOSE: Prior studies had suggested that Akt activity is elevated in a subset of breast cancers. In this study, to test the effect of active Akt-3 on estrogen receptor function, we have produced MCF-7 cells, which express active Akt-3 and examined the estrogen responsiveness of these cells in vivo and in vitro. Experimental Design: MCF-7 cells expressing active Akt-3 were studied for estradiol (E2) responsiveness in vitro by both using an estrogen receptor element reporter construct as well as looking at induction of endogenous genes. These cells were also studied in vivo after injection into nude, ovariectomized mice by following tumor growth rates in the presence or absence of E2, tamoxifen, or the pure antiestrogen, ICI 182,780 (fulvestrant). RESULTS: Akt-3-expressing cells were found to produce tumors in mice in the absence of E2 that were approximately equivalent in size to control cells in mice given E2. Moreover, the formation of tumors by the Akt-3 cells was greatly suppressed by E2, stimulated by tamoxifen, and unaffected by ICI 182,780. In the in vitro assays for gene induction by E2, the Akt-3-expressing cells exhibited similar E2 and tamoxifen responsiveness as the control cells. CONCLUSIONS: These results indicate that expression of active Akt-3 in MCF-7 cells results in E2-independent tumor growth. Moreover, the growth of these tumors is inhibited by E2 and enhanced by tamoxifen. Finally, these tumors are resistant to ICI 182,780. These findings suggest that the amount of active Akt present in breast cancers may be important in the relative efficacy of different treatments.     

Protein Kinase C<Emphasis Type="Bold"> α</Emphasis> is a marker for antiestrogen resistance and is involved in the growth of tamoxifen resistant human breast cancer cells

Development of resistance to antiestrogen treatment in breast cancer patients is a serious therapeutic problem. The molecular mechanisms contributing to resistance are currently unclear; however it is known that increased activation of growth signaling pathways is involved. Protein Kinase C α (PKCα) is associated with a diverse range of cancers and is previously shown to be overexpressed in three out of four antiestrogen resistant breast cancer cell lines. In this study we investigated whether PKCα contributes to antiestrogen resistant growth. A panel of nine resistant cell lines was investigated, all of which displayed elevated levels of PKCα expression relative to parental MCF-7 cells. Stable PKCα overexpression in MCF-7 cells significantly reduced sensitivity to the antiestrogens, tamoxifen and ICI 182,780. Two resistant cell lines were chosen for further studies: tamoxifen resistant MCF-7/TAM^R-1 (TAM^R-1) and ICI 182,780 resistant MCF-7/182^R-6 (182^R-6). Treatment with the PKCα inhibitor Ro-32–0432 resulted in a preferential growth inhibition of these two cell lines relative to MCF-7 cells. Moreover, transient down-regulation of PKCα resulted in a 30–40% growth inhibition of TAM^R-1 and 182^R-6, while MCF-7 remained unaffected. Stable PKCα knock-down in TAM^R-1 using small hairpin RNA, resulted in a tamoxifen sensitive “MCF-7–like” growth phenotype, while the same approach in 182^R-6 cells did not alter their sensitivity to ICI 182,780. These results demonstrate a functional contribution of PKCα to tamoxifen resistant growth. Furthermore, our data suggest the potential for PKCα as a marker for antiestrogen resistance and as a promising therapeutic target in the treatment of tamoxifen resistant breast cancer.     

Comparison of the effects of the PI3K/mTOR inhibitors NVP-BEZ235 and GSK2126458 on tamoxifen-resistant breast cancer cells

Background

Treatment with anti-estrogens or aromatase inhibitors is commonly used for patients with estrogen receptor-positive (ER⁺) breast cancers; however resistant disease develops almost inevitably, requiring a choice of secondary therapy. One possibility is to use inhibitors of the PI3K/mTOR pathway and several candidate drugs are in development. We examined the in vitro effects of two inhibitors of the PI3K/mTOR pathway on resistant MCF-7 cells.

Results

The derived sub-lines showed increased resistance to tamoxifen but none exhibited concomitantly increased sensitivity to the PI3K inhibitors. NVP-BEZ235 and GSK2126458 acted mainly by induction of cell cycle arrest, particularly in G₁-phase, rather than by induction of apoptosis. The lines varied considerably in their utilization of the AKT, p70S6K and ERK pathways. NVP-BEZ235 and GSK2126458 inhibited AKT signaling but NVP-BEZ235 showed greater effects than GSK2126458 on p70S6K and rpS6 signaling with effects resembling those of rapamycin.

Methods

We cultured MCF-7 cells for prolonged periods either in the presence of the anti-estrogen tamoxifen (three sub-lines) or in estrogen free medium (two sub-lines) to mimic the effects of clinical treatment. We then analyzed the effects of two dual PI3K/mTOR phosphoinositide-3-kinase inhibitors, NVP-BEZ235 and GSK2126458, on the growth and signaling pathways of these MCF-7 sub-lines. The functional status of the PI3K, mTOR and ERK pathways was analyzed by measuring phosphorylation of AKT, p70S6K, rpS6 and ERK.

Conclusion

Increased resistance to tamoxifen in these MCF-7 sub-lines is not associated with hypersensitivity to PI3K inhibitors. While both drugs inhibited AKT signaling, NVP-BEZ235 resembled rapamycin in inhibiting the mTOR pathway.Key words: breast cancer, PI3K, mTOR, BEZ235, GSK2126458, estrogen receptor, MCF-7     

Essiac® and Flor-Essence® herbal tonics stimulate the in vitro growth of human breast cancer cells

SummaryBackground People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence^? and Essiac^? Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence^? and Essiac^? for their effects on the growth of human tumor cells in culture.Methods The effect of Flor-Essence^? and Essiac^? herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors. Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay.Results Flor-Essence^? and Essiac^? herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls in both estrogen receptor positive (MCF-7 and T47D) and estrogen receptor negative (MDA-MB-231 and MDA-MB-436) cell lines. Exposure to the tonics also produced a dose-dependent increase in ER dependent luciferase activity in MCF-7 cells. A 10⁻⁷ M concentration of ICI 182,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence^? and Essiac^?, but did not affect cell proliferation.Conclusion Flor-Essence^? and Essiac^? Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action.     

Tamoxifen and ICI 182,780 increase Bcl-2 levels and inhibit growth of breast carcinoma cells by modulating PI3K/AKT, ERK and IGF-1R pathways independent of ERα

We recently showed that estrogen withdrawal from the ERα⁺, high Bcl-2-expressing breast carcinoma cells (MCF-7B) reduced Bcl-2 protein levels while increasing cell–cell adhesion, and junction formation. Here we compared these cells with the ERα⁺ and low Bcl-2-expressing MCF-7 cells and with the normal mammary epithelial cell line MCF-10-2A not expressing ERα or Bcl-2. All cell lines expressed normal HER2. Antiestrogen (Tamoxifen and ICI 182,780) treatment increased Bcl-2 levels in both MCF-7 and -7B cells and led to the formation of acinar structures. This treatment led to the dissociation of junctions and redistribution of junctional components to the cytoplasm in MCF-10-2A and -7 cells, while in MCF-7B cells junctional proteins redistributed to membranes. Antiestrogen treatment decreased PI3K/Akt activation and increased ERK activation regardless of ERα status. IGF-1R was inactivated in the antiestrogen-treated MCF-7 cells while it was activated in MCF-7B cells. Our data show that Tamoxifen and ICI 182,780 can induce growth inhibitory effects via the sustained activation/inactivation of signaling pathways that regulate cell survival, cell death and differentiation in the absence of ERα. Furthermore, Bcl-2 overexpression may alter the functional interactions among these pathways in response to antiestrogens, which also may provide a potential explanation for the observation that Bcl-2 overexpressing tumors have a better prognosis.     

Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: Comparison of immunocytochemical data with biochemical measurements

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF-7 cells under estrogenic and anti-estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER-EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E₂ and DES) and pure anti-estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4-OH-TAM and RU 39,119). E₂-induced ER down-regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down-regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre-labeling of the cells with [³H]TAZ indicated that all investigated ligands eliminate pre-existing ER through binding to newly synthetized receptors, since [³H]TAZ co-valently associates with ER; E₂ and RU 58,668 were more effective than 4-OH-TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre-exposed to [³H]TAZ. Nuclear location of [³H]TAZ-ER complexes may explain this discrepancy, since pure AE-ER complexes were reported to be incapable of nuclear translocation. Int. J. Cancer 78:760–765, 1998. © 1998 Wiley-Liss, Inc.     

(-)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells

Background: (−)-Epigallocatechin-3-gallate (EGCG) is the most active catechin present in green tea, demonstrated to have chemopreventive action and to kill cancer cells selectively. As a previous study found that catechins could compete with 17-β-estradiol for binding to estrogen receptor alpha (ERα), we asked whether EGCG could regulate ERα action. Methods: We used MCF-7, a breast carcinoma cell line having a high level of ERα expression. The cells were treated with various EGCG concentrations and cell viability was evaluated by MTT assay. ERα and pS2 expression were analyzed by RT-PCR after RNA extraction. To better define EGCG action in relation to ERα, we studied EGCG cytotoxicity on MCF-7 resistant to tamoxifen (MCF-7tam), MCF-7 treated with 10⁻⁷ M ICI 182,780 for 8 days and on MDA-MB-231, a cell line that lacked ERα by flow cytometry (FCM). Results: Both ERα and pS2 mRNA were expressed in samples treated with low EGCG concentration (30 μg/ml). At this concentration, no cell change was detectable. In contrast, pS2 expression was lost in samples treated with 100 μg/ml EGCG for 24 h, indicating ERα alteration. EGCG cytotoxicity was lower when ERα was not present (MDA-MB-231) or inactivated (by tamoxifen or ICI 182,780). Conclusions: Functionally active ERα may have a role in EGCG cytotoxicity, increasing the sensitivity to the drug. As higher EGCG concentrations also killed cells resistant to tamoxifen or treated by 10⁻⁷ M ICI 182,780, EGCG ought to be better investigated in breast carcinoma cells treated with drugs targeted to steroid receptors, as a potential complement of therapy.