Human chorionic gonadotropin (hCG) prevents the transformed phenotypes induced by 17 β-estradiol in human breast epithelial cells

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 β-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland.     

The effect of physiological concentrations of sex hormones,insulin, and glucagon on growth of breast and prostate cells supplemented with unmodified human serum

The majority of cell culture studies have assessed the effect of hormones on cancer cell growth using media supplemented with charcoal-treated fetal bovine serum (CTS). We aimed to determine whether using a system more reflective of the human condition by changing the charcoal-treated serum to an untreated pooled human serum (PHS) resulted in the same hormone responses in breast and prostate cell lines. MCF-7 breast cancer, MCF-10A non-transformed breast, and LNCaP prostate cancer cell lines supplemented with PHS were treated with high and low physiological concentrations of six hormones (17β-estradiol, dehydroepiandosterone (DHEA), dihydrotestosterone (DHT), testosterone, insulin, and glucagon). Cell growth was measured after 72 h of incubation. All hormones stimulated growth of MCF-7 cells (p < 0.05). MCF-10A cell growth was inhibited by DHEA, DHT, and testosterone (p < 0.05), unaffected by 17β-estradiol and glucagon, and stimulated by insulin (p < 0.05). LNCaP cell growth was stimulated by the highest concentration of DHEA and DHT (p < 0.05) and inhibited by the highest concentration of 17β-estradiol (p < 0.05), while insulin and testosterone, had no effect. Overall, PHS lowered the magnitude of the effect of hormones on cell growth in comparison to CTS. Due to the presence of all serum constituents, our model represents a more appropriate physiological environment for determining the effect of hormones on cancer cell growth. Further studies are required to determine the mechanisms by which added hormones interact with the constituents of untreated human serum.     

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na⁺-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na⁺. Although taurine uptake was reduced in Cl^- free medium a significant portion of taurine uptake persisted in the presence of NO₃ ^-. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the V_max of influx was increased without affecting the K_m. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na⁺. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na⁺-dependent taurine uptake may not be strictly dependent upon extracellular Cl^-.     

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G₂/M phases, whereas the G₁ phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G₂/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G₂/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G₂/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.     

A bifunctional platinum(II) antitumor agent that forms DNA adducts with affinity for the estrogen receptor

A strategy is described for the re-design of DNA damaging platinum(II) complexes to afford elevated toxicity towards cancer cells expressing the estrogen receptor (ER). Two platinum-based toxicants are described in which a DNA damaging warhead, [Pt(en)Cl₂] (en, ethylenediamine), is tethered to either of two functional groups. The first agent, [6-(2-amino-ethylamino)-hexyl]-carbamic acid 2-[6-(7α-estra-1,3,5,(10)-triene)-hexylamino]-ethyl ester platinum(II) dichloride ((Est-en)PtCl₂), terminates in a ligand for the ER. The second agent is a control compound lacking the steroid; this compound, N-[6-(2-amino-ethylamino)-hexyl]-benzamide platinum(II) dichloride ((Bz-en)PtCl₂)), terminates in a benzamide moiety, which lacks affinity for the ER. Using a competitive binding assay, Est-en had 28% relative binding affinity (RBA) for the ER as compared to 17β-estradiol. After covalent binding to a synthetic DNA duplex 16-mer, the compound retained its affinity for the ER; specificity of the binding event was demonstrated by the ability of free 17β-estradiol as a competitor to disrupt the DNA adduct-ER complex. The (Est-en)PtCl₂ compound showed higher toxicity against the ER positive ovarian cancer cell line CAOV3 than did the control compound. (Est-en)PtCl₂ was also more toxic to the ER positive breast cancer line, MCF-7, than to an ER negative line, MDA-MB231.     

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.     

The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17β-estradiol on ASMC proliferation and the mechanisms involved.Cell proliferation was estimated using the methyl-[³H]thymidine incorporation and Cell Titer 96^® AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 μM) or 17β-estradiol (1 pM-1 μM), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 μM) or wortmannin (1 μM), or the MAPK inhibitors PD98059 (100 μM) or U0126 (1 μM).After 24 h of incubation, testosterone and 17β-estradiol increased methyl-[³H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17β-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126.In conclusion, testosterone and 17β-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females.     

Dual effects of isoflavonoids from Pueraria lobata roots on estrogenic activity and anti-proliferation of MCF-7 human breast carcinoma cells

Pueraria lobata root (PLR), well known as Kudzu root, has recently become commercially available in Western dietary supplements for menopausal symptoms. The scientific basis for its action has been attributed to the action of phytoestrogens. This study aimed to investigate the estrogen-like activity of isoflavonoids isolated from P. lobata root and their safety with respect to their effect on breast cancer cell proliferation. In an E-screen assay, crude MeOH extract of PLR significantly increased the proliferation of MCF-7 cells in a concentration-dependent manner. Among the four fractions obtained by solvent fractionation of MeOH extract, the n-BuOH fraction had significant estrogen-like activities at all concentrations tested. Phytochemical analysis of the n-BuOH fraction led to the isolation of 10 isoflavones (1–10), among which genistein (10) had significant estrogen-like activities at all concentrations tested. These activities were significantly enhanced by treatment with genistein and 17β-estradiol compared with 17β-estradiol alone, and this effect was mediated by decreased expression of estrogen receptor (ER)α and phospho-ERα in MCF-7 cells. In a cell cytotoxicity assay, genistein (10) exhibited significant cytotoxicity in both ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. This cytotoxicity was characterized by the induction of apoptotic cells stained with annexin V conjugated with Alexa Fluor 488 and involved activation of mitochondria-independent and -dependent apoptosis pathways in MCF-7 cells. Our results demonstrated that genistein (10) has estrogen-like effects dependent on ER pathway activation and anti-proliferative effects mediated by the apoptosis pathway rather than the ER pathway in MCF-7 breast cancer cells.     

DHEAS prevents pro-metastatic and proliferative effects of 17ß-estradiol on MCF-7 breast cancer cells

It is generally assumed that circulating dehydroepiandrosterone sulfate (DHEAS) can be desulfated and further metabolized to estrogen, which is of concern for all patients with estrogen-responsive breast cancer. We addressed this issue by comparing the effects of DHEAS, its desulfated form DHEA, and 17ß-estradiol on human metastatic, estrogen-responsive MCF-7 breast cancer cells.Physiological concentrations of DHEAS promoted phosphorylation of Erk1/2, whereas DHEA and 17ß-estradiol failed to stimulate Erk1/2 phosphorylation, indicating that the sulfated steroid acts as an autonomous hormone. Exposure of MCF-7 cells to 17ß-estradiol stimulated cell proliferation and the expression of pro-metastatic and pro-invasive elements such as claudin-1, matrix metalloproteinase 9 (MMP9), and the CC chemokine ligand 2 (CCL2). In contrast, treatment with DHEAS did not stimulate these responses but prevented all of the actions of 17ß-estradiol, and as a consequence cell migration and invasion were completely inhibited.The results of this study not only challenge the assumption that DHEAS poses a danger as an endogenous source of estrogen, they rather favor the idea that keeping DHEAS levels within a physiological range might be supportive in treating estrogen-responsive breast cancer.     

Changes in protein tyrosine phosphatase type IVA member 1 and zinc finger protein 36 C3H type-like 1 expression demonstrate altered estrogen and progestin effect in medroxyprogesterone acetate-resistant and estrogen-independent breast cancer cell models

Estrogen stimulates proliferation in hormone-responsive breast cancer cells. Progestins inhibit the estrogen-mediated growth in these cells and are used in the treatment of mammary carcinomas. We applied cDNA microarray and real-time RT-PCR methods to reveal 17β-estradiol- and medroxyprogesterone acetate (MPA)-regulated genes in MCF-7 breast cancer cells. We identified six genes, two of which were novel MPA and/or 17β-estradiol-regulated genes: protein tyrosine phosphatase type IVA, member 1 (PTP4A1) and zinc finger protein 36, C3H type-like 1 (ZFP36L1). PTP4A1 expression was upregulated by 17β-estradiol and this was opposed by MPA treatment of the cells. ZFP36L1 proved to be a direct target of MPA. Since MPA has also been shown to bind to glucocorticoid- and androgen receptors, we studied the regulation of the genes with progesterone, synthetic progestin R5020, dexamethasone and dihydrotestosterone. We also assessed the expression and hormonal regulation of PTP4A1 and ZFP36L1 mRNA in MCF-7-derived MPA-resistant and estrogen-independent sublines. The regulation of PTP4A1 expression upon 17β-estradiol and combined 17β-estradiol and MPA treatment was completely reversed in the estrogen-independent and MPA-resistant sublines, respectively. This study suggests an important role for PTP4A1 in cell growth, and shows that MPA may potentiate the effect of 17β-estradiol in the MPA-resistant breast cancer cells.     

Inhibitory effect of estrogen on Rac1-expression in monocytes

Recruitment of circulating monocytes into the vasculature and release of reactive oxygen species (ROS) promote atherogenesis. Rac1-GTPase is an essential component of the superoxide-producing NADPH-oxidase complex. Estrogens inhibit production of vascular reactive oxygen species.Angiotensin II as well as overexpression of the constitutively active mutant RacL61 increased ROS production in monocytes. AngII-mediated ROS release was completely inhibited by overexpression of the dominant negative mutant RacN17 or treatment with 17β-estradiol. 17β-Estradiol reduced Rac1-expression concentration- and time-dependently and decreased basal, as well as AngII-induced Rac1 activity. The effects of 17β-estradiol were receptor-mediated. In vivo, down-regulation of Rac1 by 17β-estradiol was observed in human mononuclear cells of women with elevated 17β-estradiol levels after controlled ovarian hyperstimulation.In summary, the data show that down-regulation of Rac1-GTPase contributes to the inhibition of angiotensin II-mediated superoxide release by 17β-estradiol in monocytes.     

l-Leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter

The transport of l-leucine by two human breast cancer cell lines has been examined. l-Leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na⁺-independent pathway. l-Leucine uptake by both cell lines was inhibited by l-alanine, d-leucine and to a lesser extent by l-lysine but not by l-proline. Estrogen (17β-estradiol) stimulated l-leucine uptake by MCF-7 but not by MDA-MB-231 cells. l-Leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on l-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 °C. There was, however, a significant efflux of l-leucine under zero-trans conditions which was also temperature-sensitive. l-Glutamine, l-leucine, d-leucine, l-alanine, AIB and l-lysine all trans-stimulated l-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, d-alanine and l-proline had little or no effect. The anti-cancer agent melphalan inhibited l-leucine uptake by MDA-MB-231 cells but had no effect on l-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for l-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc.     

Downregulation of CXXC Finger Protein 4 Leads to a Tamoxifen-resistant Phenotype in Breast Cancer Cells Through Activation of the Wnt/β-catenin Pathway

Tamoxifen is a successful endocrine therapy drug for estrogen receptor–positive (ER+) breast cancer. However, resistance to tamoxifen compromises the efficacy of endocrine treatment. In the present study, we identified potential tamoxifen resistance–related gene markers and investigated their mechanistic details. First, we established two ER + breast cancer cell lines resistant to tamoxifen, named MCF-7/TMR and BT474/TMR. Gene expression profiling showed that CXXC finger protein 4 (CXXC4) expression is lower in MCF-7/TMR cells than in MCF-7 cells. Furthermore, CXXC4 mRNA and protein expression are lower in the resistant cell lines than in the corresponding parental cell lines. We also investigated the correlation between CXXC4 and endocrine resistance in ER + breast cancer cells. CXXC4 knockdown accelerates cell proliferation in vitro and in vivo and renders breast cancer cells insensitive to tamoxifen, whereas CXXC4 overexpression inhibits cancer cell growth and increases tamoxifen sensitivity of resistant cells. In addition, we demonstrated that CXXC4 inhibits Wnt/β-catenin signaling in cancer cells by modulating the phosphorylation of GSK-3β, influencing the integrity of the β-catenin degradation complex. Silencing the CXXC4 gene upregulates expression of cyclinD1 and c-myc (the downstream targets of Wnt signaling) and promotes cell cycle progression. Conversely, ectopic expression of CXXC4 downregulates the expression of these proteins and arrests the cell cycle in the G0/G1 phase. Finally, the small-molecule inhibitor XAV939 suppresses Wnt signaling and sensitizes resistant cells to tamoxifen. These results indicate that components of Wnt pathway that are early in response to tamoxifen could be involved as an intrinsic factor of the transition to endocrine resistance, and inhibition of Wnt signaling may be an effective therapeutic strategy to overcome tamoxifen resistance.     

Constituents of the leaves of Pseuderanthemum carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb.

A new iridoid, 5β,6β-dihydroxyantirrhide (1) was isolated from the dried leaves of Pseuderanthemum carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb. (Acanthaceae), together with 13 known compounds, including two iridoids, linarioside and antirrhinoside; five phenylethanoids, echipuroside A, verbascoside, isoverbascoside, isomartynoside and osmanthuside B; and six flavonoids, luteolin 7-O-β-d-glucopyranoside, luteolin 7-O-rutinoside, apigenin 7-O-rutinoside, apigenin 6-C-α-l-arabinopyranosyl–8-C-β-l-arabinopyranoside, apigenin 6,8-di-C-α-l-arabinopyranoside and apigenin 6-C-β-d-xylopyranosyl–8-C-α-l-arabinopyranoside. Their chemical structures were elucidated by 1D and 2D NMR as well as HR-ESI-MS spectroscopic analysis. Some purified compounds were evaluated the acetylcholinesterase inhibition and cytotoxic activities against the HeLa cervical cancer cell line and the MCF-7 breast cancer cell line at the concentration of 100 μg/mL. Luteolin 7-O-β-d-glucopyranoside exhibited cytotoxic activities against both the HeLa cervical cancer cell line and the MCF-7 breast cancer cell line. Verbascoside and isoverbascoside showed strong cytotoxic activity against the MCF-7 breast cancer cell line. The tested compounds showed the AChE inhibitory activity fairly weak.     

Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer

Aldo-keto reductase (AKR) 1C3 (type 5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD₂ to 9α,11β-PGF₂, Δ⁴-androstenedione to testosterone, progesterone to 20α-hydroxyprogesterone, and to a lesser extent, estrone to 17β-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1 μM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17β-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor α induced proliferation. MCF-7-AKR1C3 cells also reduced PGD₂, limiting its dehydration to form PGJ₂ products. The AKR1C3 product was confirmed as 9α,11β-PGF₂ and quantified with a stereospecific stable isotope dilution liquid chromatography–mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD₂ on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor γ (PPARγ) signaling by reducing formation of 15-deoxy-Δ^12,14-PGJ₂ (15dPGJ₂).     

Mitochondrial dynamics is affected by 17β-estradiol in the MCF-7 breast cancer cell line. Effects on fusion and fission related genes

Mitochondrial dynamics, specifically fusion and fission processes, maintain mitochondria integrity and function, yet at this time, effect of estrogens on fusion and fission in breast cancer cell lines has not been studied. The aim of this study was to characterize the effect of 17β-estradiol on fusion and fission-related genes, as well as on mitochondria proliferation and function. We used MCF-7 breast cancer cell line, which is estrogen sensitive (estrogen receptor positive). Cells were grown in Dulbecco's modified Eagle medium charcoal-stripped fetal bovine serum and treated with 1nM of 17β-estradiol and with/without 100nM of ICI 182,780, a drug that caused rapid degradation of estrogen receptor. mRNA levels of fusion (mfn1, mfn2, opa1) and fission-related genes (drp1 and fis1) were examined by RT-PCR, cardiolipin content by N-acridyl-orange fluorescence and oxidative phosphorylation protein levels, as well as, the major fusion and fission related protein levels, by Western blot. mRNA expression of fusion-related genes increased after 17β-estradiol-treatment for 4h; however fis1 fission-related gene expression decreased. All these effects were not found in cells pre-treated with ICI 182,780, save for the changes in mfn-1, conferring them the effects of 17β-estradiol to estrogen receptor. The changes in protein levels were less prominent, but in the same way, than in mRNA levels, showing an increase in Mfn1 and Mfn2, as well as in Drp1, but there was no change in Fis1 protein levels. Mitochondrial biogenesis was also affected by 17β-estradiol, showing an increase in mtDNA but with no change in N-acridyl-orange fluorescence. On the whole, our results suggest an imbalance in the fusion/fission ratio, with a high fusion by 17β-estradiol-estrogen receptor action, which can affect to mitochondrial biogenesis, concretely in mitochondria proliferation. According to this information, 17β-estradiol would modify mitochondrial dynamics, biogenesis and metabolism, and thus compromise the normal development and function of mitochondria in cancer affected tissues.     

Expression profiling of the genes responding to zearalenone and its analogues using estrogen-responsive genes

To compare gene expression profiles in response to estrogen or 17β-estradiol (E₂) and a mycotoxin, zearalenone (ZEA), and its analogues (collectively termed ZEA compounds), breast cancer MCF-7 cells were treated with 10 nM of E₂ or ZEA compounds including ZEA, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol. Expression profiles for 120 estrogen-responsive genes were subjected to cluster and statistical analyses using correlation coefficients or R-values. We found that all of the ZEA compounds stimulated the growth of MCF-7 cells, as much as E₂, and showed similar expression profiles to that of E₂ (R-values ranged from 0.82 to 0.96). The effect of ZEA compounds was likely mediated by estrogen-receptor-dependent Erk1/2-signaling. These results provide clues to understand the mechanism of their estrogen-like action.