Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells

Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17beta-estradiol (E2) up-regulates PI3K in an ERalpha-dependent manner, but not ERbeta, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ERalpha-positive MCF-7 cells and ERalpha-negative MDA-MB-231 cells with 10nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP(3) level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ERalpha-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ERalpha-dependent mechanism in MCF-7 cells.     

Stimulation of catecholamine synthesis through unique estrogen receptors in the bovine adrenomedullary plasma membrane by 17beta-estradiol

Incubation of cultured bovine adrenal medullary cells with 17beta-estradiol (E(2)) (0.3-100nM) or membrane-impermeable E(2)-bovine serum albumin (100nM) acutely increased (14)C-catecholamine synthesis from [(14)C]tyrosine. The stimulatory effect of E(2) was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor. E(2) also increased tyrosine hydroxylase activity and p44/42MAPK phosphorylation, the former of which was attenuated by U0126, an inhibitor of p44/42MAPK kinase. The plasma membrane isolated from the gland showed two classes of specific binding sites of [(3)H]E(2) with apparent K(d)s of 3.2 and 106nM, and B(max)s of 0.44 and 8.5pmol/mg protein, respectively. The high-affinity binding of [(3)H]E(2) was most strongly inhibited by E(2) and phytoestrogens, and to lesser extents by other steroid hormones, while it was enhanced by ICI182,780 and environmental estrogenic pollutants. These findings suggest that E(2) acutely stimulates catecholamine synthesis via activation of p44/42MAPK through unique estrogen receptors in the plasma membrane of bovine adrenal medulla.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.     

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.     

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα⁺) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.     

8-Prenylnaringenin, inhibits estrogen receptor-alpha mediated cell growth and induces apoptosis in MCF-7 breast cancer cells

The discovery that the hop constituent 8-prenylnaringenin (8PN) shows potent estrogenic activity, higher than that of the known phytoestrogens coumestrol, genistein and daidzein, has spurred an intense activity aimed at elucidating its biological profile and its dietary relevance connected with the consumption of beer. We have investigated if 8PN can induce signal transduction pathways via rapid estrogen receptor (ER) activation. Under conditions of estrogen-dependent growth, treatment of MCF-7 human breast cancer cells with 8PN induced a rapid and transient activation of the MAP kinase Erk-1 and Erk-2, with kinetics similar to those induced by 17beta-estradiol (E2). 8PN could trigger the MAP kinase pathway via dual c-Src kinase activation and association with ERalpha. Co-treatment with the ER antagonist ICI 182,780 blocked each step of this transduction pathway, confirming its ER dependence. However, and in striking contrast with E2, 8PN could not induce the PI3K/Akt pathway, resulting in altered kinetics and levels of cyclin D1 expression. In accordance with these observations, flow cytometric and biochemical analysis showed that 8PN inhibited cell cycle progression and induced apoptosis in MCF-7 cells. Interference with an ER associated PI3K pathway is proposed as a possible mechanism underlying the inhibition of survival and proliferation of estrogen responsive cells by 8PN. Taken together, our finding show that 8PN is an interesting new chemotype to explore the biology of ERs.     

Synthesis, characterization and anti-tumor activity of moxifloxacin-copper complexes against breast cancer cell lines

Novel moxifloxacin-copper complexes were synthesized, characterized and screened for anti-proliferative and apoptosis-inducing activity against multiple human breast cancer cell lines (hormone-dependent MCF-7 and T47D as well as hormone-independent MDA-MB-231 and BT-20). The results indicated that the parent compound moxifloxacin (1) does not exert any inhibitory activity against breast cancer cell lines examined. On the other hand, the copper conjugate 2 and its nitrogen adducts 3-5 exerted growth inhibitory and apoptosis-inducing activity against breast cancer cell lines without any substantial effect on non-tumorigenic breast epithelial cells MCF-10A at equimolar concentration, suggesting a cancer cell-specific activity. BT-20 cells were more sensitive to compounds 2 and 3, while compounds 4 and 5 exerted significant anti-proliferative and apoptosis-inducing effects on T47D, MDA-MB-231 and BT-20 cell lines. Our results suggest that these novel compounds could be useful for the treatment of breast cancer in the future.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

A ferrocenyl derivative of hydroxytamoxifen elicits an estrogen receptor-independent mechanism of action in breast cancer cell lines

The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERα positive (MCF-7, T-47D, ZR-75-1) and 3 ERα negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERα. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERα positive and ERα negative cells. In the presence of ERα, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERα, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERα positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERα, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.     

Synthesis of 17beta-estradiol platinum(II) complexes: biological evaluation on breast cancer cell lines

The synthesis of a novel series of 17beta-estradiol-linked platinum(II) complexes is described. The new molecules are linked with an alkyl chain at position 16alpha of the steroid nucleus and bear a 16beta-hydroxymethyl side chain. They are made from estrone in five chemical steps with an overall yield exceeding 28%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER+ and ER-) human breast cancers. The derivatives incorporating a 2-(2'-aminoethyl)pyridine ligand displayed good activity against the cell lines particularly when the connecting arm is 10 carbon atoms long.     

Downregulation of Wilms' tumor 1 protein inhibits breast cancer proliferation

High levels of Wilms' Tumor 1 (WT1) mRNA have been correlated with poor prognosis in breast cancer patients. However, the function of WT1 protein in breast cancer is not known. We observed that the levels of WT1 protein correlated with the proliferation of breast cancer cells. When the proliferation of breast cancer cells was stimulated by 17beta-estradiol, WT1 protein expression increased. But when the proliferation of breast cancer cells was inhibited by tamoxifen or all-trans retinoic acid (ATRA), WT1 protein expression decreased. We hypothesize that WT1 protein plays a role in regulating breast cancer cell proliferation. Using liposome-incorporated WT1 antisense oligodeoxynucleotides, we found that downregulation of WT1 protein expression led to breast cancer growth inhibition and reduced cyclin D1 protein levels. These results indicate that WT1 protein contributes to breast cancer progression by promoting breast cancer cell proliferation.     

The breast cancer cells response to chronic hypoxia involves the opposite regulation of NF-kB and estrogen receptor signaling

The progression of cancer is associated with tumor's ability to outgrow the existing vasculature resulting in chronic hypoxic pressure, however the molecular mechanism of cancer cell response to chronic hypoxia is poorly understood. In this study we have analyzed the reorganization of estrogen receptor (ER) signaling in breast cancer cells under chronic hypoxia and examined the role of interrelations between ER and NF-kB signaling in cell adaptation to hypoxia. Using long-term culturing of MCF-7 breast cancer cells in hypoxia-mimetic conditions (cobalt chloride) we have established a hypoxia-tolerant subline characterized by HIF-1 hyperexpression that retained the tolerance to hypoxia even when the cells were returned to normoxic conditions.The hypoxia-tolerant cells were characterized by non-affected ER signaling, irreversible suppression of NF-kB activity, and increased sensitivity to cytokine-induced apoptosis. Estradiol treatment suppressed the NF-kB activity in both parent and hypoxia-tolerant MCF-7 cells. In contrast to MCF-7 cells, the exposure of estrogen-independent MCF-7/T2 subline to chronic hypoxia was not accompanied by noticeable changes in NF-kB activity or cell sensitivity to cytokines. Taken together, the results presented demonstrate the importance of interrelations between ER and NF-kB signaling in the response of estrogen-dependent breast cancer cells to chronic hypoxia.     

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.     

Identification of membrane progestin receptors in human breast cancer cell lines and biopsies and their potential involvement in breast cancer

Novel membrane progestin receptors (mPRs) coupled to G proteins recently identified in several species, including humans, are potential intermediaries in rapid, nongenomic progestin actions observed in a wide variety of tissues. Here we demonstrate mPR mRNA and protein expression and specific membrane-associated progestin binding in MCF-7 and SK-BR-3 human breast cancer cells. Interestingly, human mPRalpha mRNA expression was higher in breast tumor biopsies than in normal tissue from the same breast. Recent studies indicate intracellular signaling pathways initiated by the mPRs are broadly similar to those induced during breast cancer growth and development. Taken together these results suggest a potential involvement of mPRs during the development or progression of breast cancer.     

Association between estradiol, estrogen receptors, total lipids, triglycerides, and cholesterol in patients with benign and malignant breast tumors

This study addresses the correlation between the levels of estradiol (E₂), total lipids, triglycerides, and cholesterol in serum and tissue samples of age-matched patients with benign (40 cases; 16 were premenopausal and 24 were postmenopausal) and malignant (50 cases; 17 were premenopausal and 33 were postmenopausal) breast tumors. Estradiol levels were determined in serum and cytosol, estrogen receptors (ER) were assayed in cytosol, and total lipids, triglycerides and cholesterol were determined in serum and membrane fractions of all benign and malignant breast disease patients. Serum E₂ was significantly higher in malignant cases than benign ones (P<0.05) with a significant reduction (40%) in postmenopausal than premenopausal women. ER-positive tumors were significantly higher in postmenopausal women with malignant breast tumors than benign cases (P<0.05). Tissue levels of total lipids, triglycerides, and cholesterol were highly significantly increased in breast cancer women than women with benign breast diseases (P<0.05, P<0.005 and P<0.05 respectively) and they were also significantly correlated with estradiol levels. It could be concluded that the uptake of lipids from plasma by the tumor tissue is greatly correlated to estradiol and it may confirm the possible role of lipids as risk factor in breast cancer.     

Estrogen receptor β in the breast: role in estrogen responsiveness and development of breast cancer

Breast cancer is one of the most common forms of cancer observed in women. Endogenous estrogen is thought to play a major role in its development and estrogen receptor blockers are the most important drugs in its treatment. It has long been thought that any conditions or exposures, which enhance estrogenic responses, would result in an increased risk for breast cancer. The discovery of the second estrogen receptor, ERβ, which can have effects opposite to those of the well-known ‘original’ estrogen receptor (now called ER) challenges this simplistic view. In order to understand breast cancer one must first understand how the normal breast is maintained. The functions of ERβ in the breast remain to be defined but from what we have learnt about its activities in in vitro systems, this estrogen receptor may have a protective role in the breast. Studies in human and rodent breasts as well as in human breast cancer biopsies reveal that ERβ is by far the more abundant of the two ERs. Despite the role of estrogen in proliferation of the breast, neither of the two ERs appears to located in epithelial cells which divide in response to estrogen. In order to define the functions of ERβ in the normal and malignant breast, we have created mice in which the ERβ gene has been inactivated. Studies of the breasts of ERβ knock out mice (BERKO) revealed abnormal epithelial growth, overexpression of Ki67 and severe cystic breast disease as mice age.     

Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines

Forskolin C₁-isoxazole derivatives (3,5-regioisomers) (11a–e, 14, 15a–h and 15, 16a–g) were synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were tested using estrogen receptor positive breast cancer cell lines MCF-7 and BT-474. Majority of the compounds exhibited activity against the p53-positive MCF-7 breast cancer cells but not against the p53-negative BT-474 breast cancer cells. Among forskolin derivatives, compounds 11a, 11c, 14a, 14f, 14g, 14h, 15b, 16g and 17b exhibited higher anti-cancer activity against MCF-7 cell line with an IC₅₀ ≤ 1 µM. The derivative 14f exhibited highest activity in both p53-positive (MCF-7) and p53-negative (BT-474) breast cancer cell lines with an IC₅₀ of 0.5 µM.     

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and -negative breast cancer cells

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-α (ER-α)-positive MCF-7 cells and ER-α-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-α transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3α, 17β-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin αvβ3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin αvβ3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-α-positive and ER-α-negative breast cancer cells, but by discrete mechanisms.