Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.     

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4′-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.     

Acteoside and martynoside exhibit estrogenic/antiestrogenic properties

Acteoside and martynoside are plant phenylpropanoid glycosides exhibiting anticancer, cytotoxic and antimetastatic activities. We investigated their potential to activate estrogen receptor isoforms ERalpha and ERbeta in HeLa cells transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha or ERbeta expression vector. Their estrogenic/antiestrogenic effects were also assessed in breast cancer cells (MCF7), endometrial cancer cells (Ishikawa) and osteoblasts (KS483), by measuring IGFBP3 levels, cell viability and number of mineralized nodules, respectively, seeking for a natural selective estrogen receptor modulator (SERM). Acteoside and martynoside antagonized both ERalpha and ERbeta (p<0.001), whereas they reversed the effect of E(2) mainly via ERalpha (p<0.001). Martynoside was a potent antiestrogen in MCF-7 cells, increasing, like ICI182780, IGFBP3 levels via the ER-pathway. In osteoblasts, martynoside induced nodule mineralization, which was abolished by ICI182780, implicating an ER-mediated mechanism. Furthermore, its antiproliferative effect on endometrial cells suggests that martynoside may be an important natural SERM. Acteoside was an antiestrogen in breast cancer cells and osteoblasts, without any effect on endometrial cells. Our study suggests that the nature is rich in selective ERalpha and ERbeta ligands, the discovery of which may lead to the development of novel neutraceutical agents.     

MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer

A search for regulators of estrogen receptor alpha (ERalpha) expression has yielded a set of microRNAs (miRNAs) for which expression is specifically elevated in ERalpha-negative breast cancer. Here we show distinct expression of a panel of miRNAs between ERalpha-positive and ERalpha-negative breast cancer cell lines and primary tumors. Of the elevated miRNAs in ERalpha-negative cells, miR-221 and miR-222 directly interact with the 3'-untranslated region of ERalpha. Ectopic expression of miR-221 and miR-222 in MCF-7 and T47D cells resulted in a decrease in expression of ERalpha protein but not mRNA, whereas knockdown of miR-221 and miR-222 partially restored ERalpha in ERalpha protein-negative/mRNA-positive cells. Notably, miR-221- and/or miR-222-transfected MCF-7 and T47D cells became resistant to tamoxifen compared with vector-treated cells. Furthermore, knockdown of miR-221 and/or miR-222 sensitized MDA-MB-468 cells to tamoxifen-induced cell growth arrest and apoptosis. These findings indicate that miR-221 and miR-222 play a significant role in the regulation of ERalpha expression at the protein level and could be potential targets for restoring ERalpha expression and responding to antiestrogen therapy in a subset of breast cancers.     

Deoxybenzoins are novel potent selective estrogen receptor modulators

Deoxybenzoins are plant compounds with similar structure to isoflavones. In this study, we evaluated the ability of two synthesized deoxybenzoins (compound 1 and compound 2) (a) to influence the activity of the estrogen receptor subtypes ERalpha and ERbeta in HeLa cells co-transfected with an estrogen response element-driven luciferase reporter gene and ERalpha- or ERbeta-expression vectors, (b) to modulate the IGFBP-3 and pS2 protein in MCF-7 breast cancer cells, (c) to induce mineralization of KS483 osteoblasts and (d) to affect the cell viability of endometrial (Ishikawa) and breast (MCF-7, MDA-MB-231) cancer cells. Docking and binding energy calculations were performed using the mixed Monte Carlo/Low Mode search method (Macromodel 6.5). Compound 1 displayed significant estrogenic activity via ERbeta but no activity via ERalpha. Compound 2 was an estrogen-agonist via ERalpha and antagonist via ERbeta. Both compounds increased, like the pure antiestrogen ICI182780, the IGFBP-3 levels. Compound 2 induced, like 17beta-estradiol, significant mineralization in osteoblasts. The cell viability of Ishikawa cells was unchanged in the presence of either compound. Compound 1 increased MCF-7 cell viability consistently with an increase in pS2 levels, whereas compound 2 inhibited the cell viability. Molecular modeling confirmed the agonistic or antagonistic behaviour of compound 2 via ER subtypes. Compound 2, being an agonist in osteoblasts, an antagonist in breast cancer cells, with no estrogenic effects in endometrial cancer cells, makes it a potential selective estrogen receptor modulator and a choice for hormone replacement therapy.     

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.     

Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

N-Alkoxypyrazoles as biomimetics for the alkoxyphenyl group in tamoxifen

The preparation of a series of novel analogues of the selective antiestrogen tamoxifen is reported. 1Z-alkoxyphenyl group in tamoxifen has been replaced by a N-alkoxypyrazole, while functionalised phenyl groups or heteroaromatics were introduced at the 2Z-position using sequential Suzuki cross coupling of 1,2-(bis)borylpinacol 1-phenylbutene with 4- or 5-iodo-1-N,N-dimethylaminoethyl or propyl-pyrazoles. Approximately 50 tamoxifen analogues were obtained and tested in an estrogen receptor (ERalpha) affinity assay. Several compounds exhibited binding affinities 2-5-fold lower than tamoxifen. Dose-response experiments with six selected compounds were carried out using two different human breast cancer cell lines, MCF-7 and the tamoxifen resistant cell line MCF-/TAM(R)-1. Both cell lines exhibited growth inhibition upon treatment with the tamoxifen analogues. Co-treatment of the cells, with estradiol and the individual compounds, were also performed. The results indicated that the observed growth inhibitory effect was mediated by the ERalpha. Analogues of the potent antiestrogen 4-hydroxytamoxifen (4-OHT) were synthesised where the 1E-4-hydroxyphenyl was replaced by a 1-hydroxypyrazol-4-yl group. However, modest growth inhibition of MCF-7 cells was observed upon treatment with these analogues. In contrast, 1Z-, 2Z-ringclosed tamoxifen analogue (59) was shown to possess antiproliferative effects on MCF-7 and MCF-/TAM(R)-1 cells in lower doses than tamoxifen.     

Calmodulin is a selective modulator of estrogen receptors

In the search for differences between ERalpha and ERbeta, we analyzed the interaction of both receptors with calmodulin (CaM) and demonstrated that ERalpha but not ERbeta directly interacts with CaM. Using transiently transfected HeLa cells, we examined the effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W7) on the transactivation properties of ERalpha and ERbeta in promoters containing either estrogen response elements or activator protein 1 elements. Transactivation by ERalpha was dose-dependently inhibited by W7, whereas that of ERbeta was not inhibited or even activated at low W7 concentrations. In agreement with these results, transactivation of an estrogen response element containing promoter in MCF-7 cells (which express a high ERalpha/ERbeta ratio) was also inhibited by W7. In contrast, transactivation in T47D cells (which express a low ERalpha/ERbeta ratio) was not affected by this CaM antagonist. The sensitivity of MCF-7 cells to W7 was abolished when cells were transfected with increasing amounts of ERbeta, indicating that the sensitivity to CaM antagonists of estrogen-responsive tissues correlates with a high ERalpha/ERbeta ratio. Finally, substitution of lysine residues 302 and 303 of ERalpha for glycine rendered a mutant ERalpha unable to interact with CaM whose transactivation activity became insensitive to W7. Our results indicate that CaM antagonists are selective modulators of ER able to inhibit ERalpha-mediated activity, whereas ERbeta actions were not affected or even potentiated by W7.     

Identification of BCAR3 by a random search for genes involved in antiestrogen resistance of human breast cancer cells.

The antiestrogen tamoxifen is important in the treatment of hormone-dependent breast cancer, although development of resistance is inevitable. To unravel the molecular mechanisms of antiestrogen resistance, a search for involved genes was initiated. Retrovirus-mediated insertional mutagenesis was applied to human ZR-75-1 breast cancer cells. Infected cells were subjected to tamoxifen selection and a panel of resistant cell clones was established. Screening for a common integration site resulted in the identification of a novel gene designated BCAR3. Transfer of this locus by cell fusion or transfection of the BCAR3 cDNA to ZR75-1 and MCF-7 cells induces antiestrogen resistance. BCAR3 represents a putative SH2 domain-containing protein and is partly homologous to the cell division cycle protein CDC48.     

Ginsenoside Rg1 exerts estrogen-like activities via ligand-independent activation of ERalpha pathway

Ginsenoside Rg1, an active ingredient commonly found in ginseng root, was previously demonstrated to be a phytoestrogen that exerted estrogen-like activity without direct interaction with estrogen receptors (ERs) in human breast cancer (MCF-7) cells. The present study was designed to determine the molecular mechanism by which Rg1 exerted estrogenic effects. Co-incubation of MCF-7 cells with 1 microM of ER antagonist ICI182780 abolished the inductive effects of Rg1 on pS2 expression as well as ERE-luciferase activity, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. To evaluate the relative involvement of ERalpha and ERbeta in mediating the actions of Rg1, ER-negative human embryonic kidney (HEK293) cells were co-transfected with the ERE-luciferase reporter construct and either ERalpha or ERbeta construct. The results showed that Rg1 could activate ERE-luciferase activity via the ERalpha-mediated pathway in a dose-dependent manner (10(-14) to 10(-6)M); whereas, the activation of ERbeta-mediated ERE-luciferase activity by Rg1 only occur at high concentration (10(-6)M). Furthermore, the results showed that 1pM Rg1 could rapidly induce phosphorylation of the AF-1 domain of ERalpha at serine 118 residue within the first 5 min of incubation, suggesting that Rg1 activates ERalpha in a ligand-independent manner. Taken together, our results indicate that Rg1 preferentially activates ERalpha via phosphorylation of AF-1 domain in the absence of receptor binding. This study is the first to provide evidence that ginsenoside Rg1 exerts estrogen-like actions via ligand-independent activation of ERalpha pathway.     

The ERalpha/ERbeta ratio determines oxidative stress in breast cancer cell lines in response to 17Beta-estradiol

The effects of 17beta‐estradiol (E2) are mediated through activation of estrogen receptors (ER): ERalpha and ERbeta. It is known that ERalpha/ERbeta ratio is higher in breast tumors than in normal tissue. Since antioxidant enzymes and uncoupling proteins (UCPs) are reactive oxygen species (ROS) production and mitochondrial biogenesis regulators, our aim was to study the E2‐effect on oxidative stress, antioxidant enzyme expression, and UCPs in breast cancer cell lines with different ERalpha/ERbeta ratios. The lower ERalpha/ERbeta ratio T47D cell line showed low ROS production and high UCP5 levels. However, the higher ERalpha/ERbeta ratio MCF‐7 cell line showed an up‐regulation of antioxidant enzymes and UCPs, yet exhibited high oxidative stress. As a result, a decrease in antioxidant enzyme activities and UCP2 protein levels, coupled with an increase in oxidative damage was found. On the whole, these results show different E2‐effects on oxidative stress regulation, modulating UCPs, and antioxidant enzymes, which were ERalpha/ERbeta ratio dependent in breast cancer cell lines. J. Cell. Biochem. 113: 3178–3185, 2012. © 2012 Wiley Periodicals, Inc.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) > MCF-7 (57%)>Ishikawa (51%) > HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.     

Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein

Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha-positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells.     

Response of estrogen receptor containing tumour cells to pure antiestrogens and the calmodulin inhibitor, calmidzolium chloride

Cell survival is dependent on both external and internally generated signalling processes and current strategies for medical intervention in neoplastic disease are directed towards signal transduction blockade. Redundancy in signalling pathways may mean, however, that a combination of agents is required for the maximal therapeutic benefit. We have explored this idea with regard to the antiestrogen sensitivity of estrogen dependent tumours. Using estrogen receptor (ER) containing tumour cell lines, we have determined whether antiestrogens increase the cytotoxicity of the potent calmodulin inhibitior, calmidzolium chloride (CCl). For the pituitary tumour cell line GH(3), CCl induces a form of apoptotic cell death and co-treatment with the pure antiestrogen, ZM 182780, enhances sensitivity to the calmodulin inhibitor, by at least two fold. In contrast to the pure steroidal antiestrogens, the triphenylethylenes, tamoxifen and 4-hydroxytamoxifen give no enhancing effect on CCl induced cell death. Although CCl induces apoptosis of several ER containing breast cancer cell lines, unlike the pituitary tumour cells, ZM 182780 is unable to increase their sensitivity to calmodulin inhibition. Further studies strongly suggest that cell death in response to calmodulin inhibition is the result of metabolic disruption and that for GH(3) cells, this is enhanced by antiestrogen treatment.