ER-positive Human Breast Cancer Cell Research Articles

Puerarin Exerted Anti-Osteoporotic Action Independent of Estrogen Receptor-Mediated Pathway

Puerarin, a daidzein-8-C glucoside, is the major isoflavonoid in Kudzu (Pueraria lobata), and is unique in that it contains C-C conjugated glucose at position 8 of the isoflavonoid structure. A puerarin diet at a dose of 5 mg/kg b.w./d to fed ovariectomized mice for 2 mo diminished the urinary deoxypyridinoline, a typical bone-degradation product. Since the bone absorption marker, serum tartarate-resistant acid phosphatase (TRAP) activity of puerarin-fed mice decreased but the bone formation marker, osteocalcin level, did not alter, the puerarin diet was proved to specifically depress the bone absorption, but not the overall bone metabolism. In accordance with that results, the femur structure of puerarin-fed mice was restored compared with that of puerarin-free diet mice. The atrophied uterine due to low estrogen (E2) level after ovariectomy was not restored by the puerarin diet, suggesting that puerarin exerted the anti-osteoporotic action through a non estrogen receptor (ER) mediated-pathway, in vivo. The growth of an ER-positive human breast cancer cell, MCF-70, was not enhanced by puerarin, suggesting that puerarin did not show estrogen-like action on MCF-7 cells, even at a ten thousand times higher concentration than that of E2. Furthermore, ICI182,780 (ICI), an estrogen antagonist, suppressed the enhanced growth of MCF-7 cells by E2, but not that by puerarin. In an ER-binding assay, puerarin was proved not to bind to ERα or β, or if all, extremely weakly, although daidzein, an aglycon of puerarin, showed a little stronger binding compared with puerarin. All these results strongly indicate that puerarin exerts its anti-osteoprotic action independently of the ER-mediated pathway.

P4-01-17: TIMP-1 Over-Expression Confers Resistance of MCF-7 Breast Cancer Cells to Fulvestrant.

Abstract Background Endocrine resistance represents a major challenge in the management of estrogen receptor (ER) positive breast cancer. Currently no predictive biomarkers for endocrine resistance in ERpositive breast cancer patients are in clinical use. In a clinical study, patients with metastatic breast cancer and high levels of serum Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) had less benefit from endocrine therapy than patients with a lower level of serum TIMP-1 [1]. Therefore, we evaluated the association between TIMP-1 and response to endocrine therapy using an in vitro approach. We have previously presented initial results on TIMP-1 and response to endocrine therapy [2]. Materials and Methods: MCF-7 cells were stably transfected with pcDNA3.1(Hyg)-TIMP-1 plasmid, and a panel of 11 subclones with different expression levels of TIMP-1 was generated. TIMP-1 expression levels were confirmed using enzyme-linked immunosorbent assay (ELISA). Four subclones with high or low TIMP-1 expression were analyzed for the growth response to estrogen, 4-hydroxytamoxifen and fulvestrant. These four subclones were analyzed for protein expression by western blotting. All subclones were analyzed by whole human genome oligo microarrays 4×44K for determination of gene expression levels. Data were analyzed using the limma R/Bioconductor package. Paired-end Solexa sequencing was applied to selected subclones with high and low TIMP-1 levels to identify transcriptomic changes. Results: High expression of TIMP-1 was associated with resistance to fulvestrant, whereas growth response to either estrogen or 4-hydroxytamoxifen was independent of TIMP-1 expression levels. High expression of TIMP-1 protein and mRNA was associated with undetectable levels of progesterone receptor (PgR) protein and mRNA whereas ER protein and mRNA levels were unaffected by TIMP-1. To characterize the potential role of TIMP-1 in estrogen signaling we analyzed the expression of reported estrogen-responsive genes and no general change was observed. We identified genes that correlated positively or negatively to TIMP-1 expression. Among the identified genes was PgR, which is a direct target for ER. Conclusion: Our data suggest that a high expression of TIMP-1 in vitro is associated with resistance to fulvestrant but not to 4-hydroxytamoxifen. Estrogen-regulated genes are not generally affected by changes in TIMP-1 expression levels and therefore TIMP-1 appears to affect endocrine resistance through other mechanisms than globally regulating ER signaling. However, high expression of TIMP-1 is associated with loss of PgR and this may be related to the resistance towards fulvestrant.

ShRNA-mediated gene silencing of MTA1 influenced on protein expression of ER alpha, MMP-9, CyclinD1 and invasiveness, proliferation in breast cancer cell lines MDA-MB-231 and MCF-7 in vitro

BackgroundMTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA) expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation.MethodsShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.ResultsDown-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05). However, after inhibiting mRNA levels of MTA1, protein expression of ER alpha, MMP-9, cyclinD1 and the changes of cancer cells invasiveness, proliferation, cells cycle were no statistical difference in ER-positive human breast cancer cell lines MCF-7 (P > 0.05).ConclusionsShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.

Is Soy Consumption Good or Bad for the Breast?1–3

Genistein in soy activates estrogen receptor (ER)-α and ERβ and acts as an estradiol in multiple target tissues. Because estrogens increase breast cancer risk and genistein promotes the growth of ER-positive human breast cancer cells, it has remained unclear whether this isoflavone or soy is safe. Results reviewed here suggest that women consuming moderate amounts of soy throughout their life have lower breast cancer risk than women who do not consume soy; however, this protective effect may originate from soy intake early in life. We also review the literature regarding potential risks genistein poses for breast cancer survivors. Findings obtained in 2 recent human studies show that a moderate consumption of diet containing this isoflavone does not increase the risk of breast cancer recurrence in Western women, and Asian breast cancer survivors exhibit better prognosis if they continue consuming a soy diet. The mechanisms explaining the breast cancer risk-reducing effect of early soy intake or the protective effect in Asian breast cancer survivors remain to be established. We propose that the reduction in risk involves epigenetic changes that result in alterations in the expression of genes that regulate mammary epithelial cell fate, i.e. cell proliferation and differentiation. Lifetime soy consumption at a moderate level may prevent breast cancer recurrence through mechanisms that change the biology of tumors; e.g. women who consumed soy during childhood develop breast cancers that express significantly reduced Human epidermal growth factor receptor 2 levels. More research is needed to understand why soy intake during early life may both reduce breast cancer risk and risk of recurrence.

Of Mice and (Wo)Men: Is This Any Way to Test a New Drug?

Of Mice and (Wo)Men: Is This Any Way to Test a New Drug?

Novel identification of UDP-glucuronosyltransferase 1A10 as an estrogen-regulated target gene

Recently, we have shown that UGT1A10 is actively involved in the inactivation of E 1, E 2, and their 2- and 4-hydroxylated derivatives. In the present study, we show for the first time that treatment of the MCF-7 ER-positive breast cancer cell line with E 2 produces a dose-dependent up-regulation of UGT1A10 mRNA levels, followed by a steady down-regulation. In contrast, E 2 did not stimulate mRNA expression in the MDA-MB-231 (ER)-negative breast cancer cell line. Expression of UGT1A10 mRNA was blocked by the antiestrogen, ICI 182,780, but not by the transcriptional inhibitor, actinomycin- d. These findings suggest that regulation of UGT1A10 mRNA might be a primary transcriptional response mediated through the ER. Expression of UGT1A10 mRNA was also stimulated by other estrogenic compounds including propylpyrazoletriol (PPT) and genistein (Gen). Exposure of MCF-7 cells to 0.1 nM E 2 up-regulated, and then down-regulated, UGT1A protein and enzymatic activity toward E 2 at 10 nM E 2 as determined by Western blot and glucuronidation activity assays. Collectively, these results suggest that induction of UGT1A10 mRNA expression by E 2 might be mediated through ER, and that this isoform is a novel, estrogen-regulated target gene in MCF-7, ER-positive human breast cancer cells. The finding of E 2-induced expression of UGT1A10 mRNA, followed by the down-regulation of UGT1A10 at pharmacological concentrations of E 2, might have a significant moderating effect on E 2 availability for ER and estrogen clearance, thereby promoting the signaling of E 2 in breast cancer cells.

A novel therapeutic strategy for ER-negative human breast cancers, targeting AP-1 activity

14129 Background: While agents targeting estrogen receptors are the most effective in adjuvant therapy for human breast cancers expressing estrogen receptor(ER), breast cancers lacking ER are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Although a transcription factor, AP-1, is known to be related to tumor malignancy including metastasis, invasion and drug-resistance, it remains to be elucidated how AP-1 plays in development and expression of malignant characters of human breast cancers. Methods and Results: Here, we used MX-1, a human breast cancer cell line lacking ER and several ER positive cell lines, to clarify the roles of AP-1 and the therapeutic efficacy of ascochlorin, a newly developed prenylphenol antibiotic on ER-negative breast cancer. We found that MX-1 exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos and Fra-1 when compared with conventional ER-positive human breast cancer cell lines. Consistent with this study in vitro, histological study on human breast cancer tissues suggests that ER-negative cancers express high Fra-1 protein, and that paclitaxel- sensitive cancers express low Fra-1 protein. The ascochlorin, which inhibits AP-1 through the Erk signaling pathway, suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Moreover, administration of ascochlorin elongated life span of mice intraperitoneally implanted with murine mammary carcinoma cells. Conclusions: Our results suggest that AP-1 is an effective clinical target molecule for the treatment of ER-negative human breast cancer, and that ascochlorin is promising therapeutic agent for these refractory breast cancers. No significant financial relationships to disclose.

Estrogen-Regulated Gene Networks in Human Breast Cancer Cells: Involvement of E2F1 in the Regulation of Cell Proliferation

Estrogens generally stimulate the proliferation of estrogen receptor (ER)-containing breast cancer cells, but they also suppress proliferation of some ER-positive breast tumors. Using a genome-wide analysis of gene expression in two ER-positive human breast cancer cell lines that differ in their proliferative response to estrogen, we sought to identify genes involved in estrogen-regulated cell proliferation. To this end, we compared the transcriptional profiles of MCF-7 and MDA-MB-231ER+ cells, which have directionally opposite 17beta-estradiol (E2)-dependent proliferation patterns, MCF-7 cells being stimulated and 231ER+ cells suppressed by E2. We identified a set of approximately 70 genes regulated by E2 in both cells, with most being regulated by hormone in an opposite fashion. Using a variety of bioinformatics approaches, we found the E2F binding site to be overrepresented in the potential regulatory regions of many cell cycle-related genes stimulated by estrogen in MCF-7 but inhibited by estrogen in 231ER+ cells. Biochemical analyses confirmed that E2F1 and E2F downstream target genes were increased in MCF-7 and decreased in 231ER+ cells upon estrogen treatment. Furthermore, RNA interference-mediated knockdown of E2F1 blocked estrogen regulation of E2F1 target genes and resulted in loss of estrogen regulation of proliferation. These results demonstrate that regulation by estrogen of E2F1, and subsequently its downstream target genes, is critical for hormone regulation of the proliferative program of these breast cancer cells, and that gene expression profiling combined with bioinformatic analyses of transcription factor binding site enrichment in regulated genes can identify key components associated with nuclear receptor hormonal regulation of important cellular functions.

Stimulatory effects of extract prepared from the bark of Cinnamomum cassia blume on the function of osteoblastic MC3T3‐E1 cells

The ethanol extract from the bark of Cinnamomum cassia Blume (CCE) was tested for estrogenic activity. CCE (4-60 microg/mL) significantly induced the growth of MCF-7 cells, an ER-positive human breast cancer cell line, over that of untreated control cells (p < 0.05). In the ER competitive binding assay, CCE showed higher affinity with ERbeta compared with ERalpha. To investigate the bioactivities of CCE, which act on bone metabolism, the effects of CCE on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were studied. CCE (4-60 microg/mL) dose-dependently increased the survival of MC3T3-E1 cells. In addition, CCE (10 and 50 microg/mL) increased alkaline phosphatase (ALP) activity, collagen synthesis and osteocalcin secretion in MC3T3-E1 cells. Treatment with CCE (10 and 50 microg/mL) prevented apoptosis induced by TNF-alpha (10(-10) m) in osteoblastic cells. In the presence of TNF-alpha, culture with CCE (10-100 microg/mL) for 48 h inhibited the production of IL-6 and nitric oxide in osteoblastic MC3T3-E1 cells. These results suggest that Cinnamomum cassia has a direct stimulatory effect on bone formation in vitro and may contribute to the prevention of osteoporosis and inflammatory bone diseases.

Polyamine Analogues Down-regulate Estrogen Receptor α Expression in Human Breast Cancer Cells

The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor alpha (ERalpha) and ERalpha target genes in ER-positive human breast cancer cell lines, whereas neither ERbeta nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERalpha in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERalpha transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERalpha minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine-inhibited ERalpha expression, suggesting that AP-1 is a positive regulator of ERalpha expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERalpha induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells.

Effect of Estrogen Receptor (ER) on Benzo[a]pyrene–DNA Adduct Formation in Human Breast Cancer Cells

To investigate the role of estrogen and estrogen receptor (ER) during benzo[a]pyrene (BaP) carcinogenesis, BaP–DNA adduct formation, and DNA synthesis were examined in ER-positive, MCF-7, and ER-negative, MDA-MB-231, human breast cancer cell lines. In MCF-7, the ER-positive human breast cancer cell line, treated with BaP, the formation of BaP–DNA adducts and DNA synthesis were inhibited in a concentration-responsive manner, but there was no change in MDA-MB-231, the ER-negative cell line. In the [3H]BaP–DNA binding assay, an increase of BaP–DNA adduct formation was observed with 17β-estradiol (E2)-induced ERα. Treatments of [3H]BaP in conjunction with the E2 induced a 2.1-fold increase in BaP–DNA adduct over BaP alone in the ER-positive MCF-7 cell line. In addition, the antiestrogen tamoxifen (TAM) blocked this effect by 82%, while E2 produced no change in the ER-negative MDA-MB-231 cell line. These observations suggest that the increased formation of BaP–DNA adducts may be mediated through the ERα expressed by E2. This research was supported by the Brain Korea 21 project 2003.

Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells

We compared in this study the effects of 2-methoxyestradiol (2-MeO-E 2) on the growth of two estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and two ER-positive human breast cancer cell lines (MCF-7 and T-47D). 2-MeO-E 2 exerted a concentration-dependent antiproliferative action in the ER-negative MDA-MB-231 and MDA-MB-435s cells. The presence or absence of exogenous 17β-estradiol (E 2) in the culture medium did not affect the potency and efficacy of 2-MeO-E 2’s antiproliferative action in these ER-negative cells. When the ER-positive MCF-7 and T-47D cells were cultured in a medium supplemented with 10 nM of exogenous E 2, 2-MeO-E 2 at 750 nM to 2 μM concentrations exerted a similar antiproliferative effect. However, when the ER-positive cell lines were cultured in the absence of exogenous E 2, 2-MeO-E 2 at relatively low concentrations (10–750 nM) had a moderate mitogenic effect, with its apparent efficacy 75–80% of that of E 2. This mitogenic effect of 2-MeO-E 2 was ER-mediated and largely attributable to 2-MeO-E 2’s residual estrogenic activity on the basis of our following findings: (i) its effect was only manifested in the ER-positive cells but not in the ER-negative cells; (ii) its effect in the ER-positive cells was partially or fully abolished when exogenous E 2 was concomitantly present in the culture medium; (iii) 2-MeO-E 2 retained 1–2% of E 2’s binding affinity for the human ERα and ERβ, and its mitogenic effect was inhibited in a concentration-dependent manner by ICI-182,780, a pure ER antagonist; and (iv) its effect was not due to its metabolic conversion to 2-hydroxyestradiol. Our timely findings are of importance to the on-going clinical trials designed to evaluate 2-MeO-E 2’s effectiveness for the treatment of different types (ER-positive or ER-negative) of human breast cancer. This knowledge will improve the design of clinical trials as well as the interpretation of clinical outcomes when 2-MeO-E 2 is used as a single agent therapy or as part of a combination therapy for human breast cancer.

Measurement of estrogen receptors in intact cells by flow cytometry

Background Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D5. The stained cells were analyzed by flow cytometry. Results The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80–99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. Conclusions Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis. Cytometry 41:109–114, 2000 © 2000 Wiley-Liss, Inc.

Differences in uptake and metabolism of retinoic acid between estrogen receptor-positive and -negative human breast cancer cells

Our previous work had shown that retinoic acid (RA) inhibits cell growth and induces apoptosis in estrogen receptor-positive (ER-positive) MCF-7 and T-47D human breast carcinoma cells, but not in ER-negative human breast carcinoma cells MB-231 and MB-453. The purpose of this work was to determine whether these differences might be due to differences in uptake and metabolism of the drug between ER-positive and ER-negative cells. We measured RA uptake in cultured human breast cancer cells and determined its metabolism by high-pressure liquid chromatographic analysis. The two ER-positive cell lines reached maximum RA uptake at about 2 h, followed by a sharp decline, so that most RA had disappeared from the cells and from the medium by 24 h and was found as oxidation products in the culture medium. In contrast, the two ER-negative cell lines showed a pattern of lower accumulation without the sharp increase and subsequent steep decline, so that by 24 h there was more RA in these cells and their culture medium than in the RA-responsive ER-positive cells, even though at 2 h the ER-negative cells had taken up less RA than the ER-positive cells. Kinetic analysis of the uptake of RA in MCF-7 cells was consistent with rapid movement across the cell membranes and the actual rate determined by diffusion of albumin-bound retinoid to the cells. This study is the first to demonstrate profound differences in RA accumulation and confirms previous results on different rates of RA metabolism between ER-positive and ER-negative human breast cancer cells. The findings reported here, therefore, may introduce additional elements to be considered in the design of new drugs for cancer chemoprevention and therapy.

Measurement of estrogen receptors in intact cells by flow cytometry

Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry. The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.

The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells.

The effect of oestrogens on oestrogen-receptive organs and cells is mediated via intracellular receptors (ERalpha and ERbeta). Oestrogen receptor gene polymorphisms in the region encoding the N-terminal portion of the protein are reportedly associated with pathological conditions including breast cancer, hypertension, spontaneous abortion and coronary heart disease. A silent mutation in codon 10 of exon 1, detected in ER-negative and ER-positive human breast cancer cell lines, in breast tumors and blood DNA from breast cancer patients, has been recognized as a polymorphic site. In this study we examined, by denaturing gradient-gel electrophoresis and DNA sequence analysis, the possible presence of a codon 10 polymorphic site in normal oestrogen target organs and cells such as the uterus (myometrium and endometrium), in the placenta and peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected ER codon 10 polymorphism in these samples and have compared them to those observed in breast cancer samples. All tissues and cells studied were homozygous for the wild-type gene, and were heterozygous as well as homozygous for the codon-10-variant type. These results indicate that the presence of the codon-10-variant type is not a characteristic of breast cancer. Out current findings suggest that further investigations are warranted to elucidate the possible linkage of ER codon 10 polymorphism to physiological and pathological conditions.

The inhibition of the estrogenic effects of pesticides and environmental chemicals by curcumin and isoflavonoids.

Many environmental chemicals and pesticides have been found to be estrogenic and have been shown to stimulate the growth of estrogen receptor-positive (ER-positive) human breast cancer cells. Since it is difficult to avoid human exposure to environmental estrogens, a potentially important area of research is the development of dietary strategies to prevent the stimulated growth of breast tumors by environmental estrogens. In this context, the inhibitory action of curcumin and a combination of curcumin and isoflavonoids were studied in ER-positive human breast cancer cells (MCF-7 and T47D) and ER-negative MDA-MB-231 cells induced by the pesticide o,p'-DDT and the environmental pollutants 4-nonylphenol and 4-octylphenol. The median inhibitory concentration (IC50) for curcumin in T47D cells was 10 microM when measured at either a 48-hr or a 6-day incubation time. The IC50 value for curcumin was within the 8-10 microM range for inhibiting the growth of T47D cells induced by a 10- microM concentration each of 4-nonylphenol, 4-octylphenol, and o, p'-DDT. The IC50 for curcumin in MCF-7 cells induced by 10 microM of either o,p'-DDT, 4-octylphenol, or 4-nonylphenol were 9, 39, and >50 microM, respectively. A combination of curcumin and isoflavonoids was able to inhibit the induced growth of ER-positive cells up to 95%. For MDA-MB-231 cells, the IC50 for curcumin was 17 microM, which was reduced to 11 microM in the presence of 25 microM genistein. Curcumin and genistein induce drastic changes in the morphological shape of both ER-positive and ER-negative cells. Data presented here indicate that a mixture of curcumin and isoflavonoids is the most potent inhibitor against the growth of human breast tumor cells. These data suggest that combinations of natural plant compounds may have preventive and therapeutic applications against the growth of breast tumors induced by environmental estrogens.

A Pure Antiestrogen, ICI 182,780, Stimulates the Growth of Tamoxifen-Resistant KPL-1 Human Breast Cancer Cells in vivo but not in vitro

The critical mechanisms responsible for antiestrogen resistance have not yet been elucidated. We previously established a breast cancer cell line, KPL-1, derived from a patient with recurrent disease which appeared under tamoxifenadministration. In a previous study, we suggested that this cell line is estrogen receptor (ER)-positive but tamoxifen-resistant. In the present study, the effects of a pure antiestrogen, ICI 182,780, on this cell line were investigated. Although tamoxifen inhibited neither cell growth nor estradiol-stimulated transcriptional activity in vitro, ICI 182,780, significantly inhibited both of them. Tamoxifen and ICI 182,780 were then administered to female nude mice bearing KPL-1 tumors. Tamoxifen had no effect on tumor growth, but ICI 182,780 unexpectedly stimulated it (p = 0.022). Estradiol tended to inhibit tumor growth (p = 0.198). Immunohistochemical analysis revealed that ICI 182,780 significantly increased the Ki6-labeling index (p<0.001) but estradiol decreased it (p = 0.035). To explore the possible mechanisms of these phenotypes, the mRNA levels of ER-α,ER-β, transforming growth factor-β1, fibroblast growth factor (FGF)-1 and FGF-4 in KPL-1 cells were compared with those in other ER-positive human breast cancer cell lines by reverse-transcription polymerase chain reaction. FGF-1 was overexpressed only in KPL-1 cells. This cell line is the first breast cancer cell line to be growth-stimulated by ICI 182,780 in vivo. Paracrine interaction between tumor cells and stromal cells mediated by growth factors, such as FGF-1, might be a key factor to explain the unique hormone responsiveness of KPL-1 cells.

Estrogen Regulates the Association of Intermediate Filament Proteins with Nuclear DNA in Human Breast Cancer Cells

In a previous study we showed that the levels of the intermediate filament proteins, cytokeratins 8, 18, and 19, in the nuclear matrix-intermediate filament (NM-IF) fraction from the hormone-dependent and estrogen receptor (ER)-positive human breast cancer cell line T-47D5 were regulated by estrogens. In contrast, estrogens did not regulate the cytokeratins in the NM-IF fraction of the hormone-independent and ER-positive cell line, T5-PRF. In this study, human breast cancer cells were treated with cis-diamminedichloroplatinum to cross-link protein to nuclear DNA in situ, and proteins bound to DNA were isolated. We show that cytokeratins 8, 18, and 19 of T-47D5 and T5-PRF were associated with nuclear DNA in situ. The levels of the cytokeratins 8, 18, and 19 bound to nuclear DNA or associated with the cytoskeleton of T-47D5 human breast cancer cells decreased when estrogens were depleted or the pure antiestrogen ICI 164,384 was added. In contrast, the cytokeratin levels associated with nuclear DNA or cytoskeleton were not significantly affected by estrogen withdrawal or antiestrogen administration in T5-PRF cells. These observations suggest that estrogen regulates the organization of nuclear DNA by rearrangement of the cytokeratin filament network in hormone-dependent, ER-positive human breast cancer cells and that this regulation is lost in hormone-independent, ER-positive breast cancer cells.

Antitumor mechanism of action of a cyclopropyl antiestrogen (compound 7b) on human breast cancer cells in culture.

Cyclopropyl compound 7b [(Z)-1,1-dichloro-2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxyphenyl)-3-cyclopropane] has been shown to be a pure antiestrogen in mouse uterine tissue. Antitumor activity was examined by evaluating the influence of 7b on the proliferation, estrogen receptor (ER) affinity and cell-surface morphology of ER-positive and ER-negative human breast cancer cells in culture. The antiproliferative potency of 7b was found to be equal to tamoxifen in ER-positive MCF-7 human breast cancer cells. Further, the antiproliferative activities of 7b and tamoxifen were reversed by coadministration of estradiol. Accordingly, the antiproliferative activity of compound 7b appears to be estrogen-mediated since it did not influence the growth of either ER-negative MDA-MB-231 human breast cells or A-549 human lung cancer cells in culture. An ER-dependent mechanism of action is also supported by the specific binding affinity of 7b for ER in MCF-7 cells. Further, a study of cell surface morphology using scanning electron microscopy (SEM) revealed that 7b reduced the density and distribution of microvilli (MV) on MCF-7 cells, which was reversed by coadministration of estradiol. Compound 7b did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7b inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on either ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study confirm an antiestrogenic mechanism of action for 7b as previously observed in vivo and suggest that 7b would be effective in the treatment of estrogen-dependent breast cancer or as a prophylactic treatment for women with a high risk of breast cancer development.