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Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein.

ABSTRACT: The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor ? (ER?) and ? (ER?)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ER? expression (T47D-ER?), the effect of a varying intracellular ER?/ER? ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ER?-expressing T47D-ER? cells with inhibited ER? expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ER? breast cancer cells with low levels of ER? and no expression of ER?. In addition, data from our study indicate that ER?-mediated gene and protein expression counteracts ER?-mediated effects because in T47D-ER? cells expressing ER? and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ER? decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ER? cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ER?/ER? ratio) in the cells or tissue of interest.

SUBMITTER: Sotoca AM

PROVIDER: S-EPMC3013448 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein.

Sotoca Ana M AM Gelpke Maarten D Sollewijn MD Boeren Sjef S Ström Anders A Gustafsson Jan-Åke JÅ Murk Albertinka J AJ Rietjens Ivonne M C M IM Vervoort Jacques J

Molecular & cellular proteomics : MCP 20100930 1

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expres ...[more]

PMID: 20884965

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Project description:BackgroundEndocrine disrupting chemicals are widely distributed in the environment and derive from many different human activities or can also be natural products synthesized by plants or microorganisms. The phytoestrogen, genistein (4', 5, 7-trihydroxy-isoflavone), is a naturally occurring compound found in soy products. Genistein has been the subject of numerous studies because of its known estrogenic activity.Methodology/principal findingsWe report that genistein exposure of zebrafish embryos induces apoptosis, mainly in the hindbrain and the anterior spinal cord. Timing experiments demonstrate that apoptosis is induced during a precise developmental window. Since adding ICI 182,780, an ER antagonist, does not rescue the genistein-induced apoptosis and since there is no synergistic effect between genistein and estradiol, we conclude that this apoptotic effect elicited by genistein is estrogen-receptors independent. However, we show in vitro, that genistein binds and activates the three zebrafish estrogen receptors ERalpha, ERbeta-A and ERbeta-B. Furthermore using transgenic ERE-Luciferase fish we show that genistein is able to activate the estrogen pathway in vivo during larval stages. Finally we show that genistein is able to induce ectopic expression of the aromatase-B gene in an ER-dependent manner in the anterior brain in pattern highly similar to the one resulting from estrogen treatment at low concentration.Conclusion/significanceTAKEN TOGETHER THESE RESULTS INDICATE THAT GENISTEIN ACTS THROUGH AT LEAST TWO DIFFERENT PATHWAYS IN ZEBRAFISH EMBRYOS: (i) it induces apoptosis in an ER-independent manner and (ii) it regulates aromatase-B expression in the brain in an ER-dependent manner. Our results thus highlight the multiplicity of possible actions of phytoestrogens, such as genistein. This suggests that the use of standardized endpoints to study the effect of a given compound, even when this compound has well known targets, may carry the risk of overlooking interesting effects of this compound.

Project description:Pretreatment with a phytoestrogen genistein has been shown to attenuate the development of pulmonary hypertension (PH). Because PH is not always diagnosed early, we examined whether genistein could also reverse preexisting established PH and prevent associated right heart failure (RHF). PH was induced in male rats by 60 mg/kg of monocrotaline. After 21 days, when PH was well established, rats received daily injection of genistein (1 mg/kg per day) for 10 days or were left untreated to develop RHF by day 30. Effects of genistein on human pulmonary artery smooth muscle cell and endothelial cell proliferation and neonatal rat ventricular myocyte hypertrophy were assessed in vitro. Severe PH was evident 21 days after monocrotaline, as peak systolic right ventricular pressure increased to 66.35±1.03 mm Hg and right ventricular ejection fraction reduced to 41.99±1.27%. PH progressed to RHF by day 30 (right ventricular pressure, 72.41±1.87 mm Hg; RV ejection fraction, 29.25±0.88%), and mortality was ≈75% in RHF rats. Genistein therapy resulted in significant improvement in lung and heart function as right ventricular pressure was significantly reduced to 43.34±4.08 mm Hg and right ventricular ejection fraction was fully restored to 65.67±1.08% similar to control. Genistein reversed PH-induced pulmonary vascular remodeling in vivo and inhibited human pulmonary artery smooth muscle cell proliferation by ≈50% in vitro likely through estrogen receptor-β. Genistein also reversed right ventricular hypertrophy (right ventricular hypertrophy index, 0.35±0.029 versus 0.70±0.080 in RHF), inhibited neonatal rat ventricular myocyte hypertrophy, and restored PH-induced loss of capillaries in the right ventricle. These improvements in cardiopulmonary function and structure resulted in 100% survival by day 30. Genistein restored PH-induced downregulation of estrogen receptor-β expression in the right ventricle and lung. In conclusion, genistein therapy not only rescues preexisting severe PH but also prevents the progression of severe PH to RHF.

Project description:Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex, and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions.

Project description:Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 and acetate-tolerant mutant AcR at different time points of 130, 148, 166, and 190h post-inoculation with 10g/L sodium acetate (NaAc) treatment were carried out to investigate the expression differences between ZM4 and AcR. Two biological replicates for treatment or control condition.

Dataset Information

Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein.

Publications

Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein.

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