DAX-1, as an androgen-target gene, inhibits aromatase expression: a novel mechanism blocking estrogen-dependent breast cancer cell proliferation.

Sexual hormones, estrogens and androgens, determine biological response in a tissue- and gender-specific manner and have a pivotal role in endocrine-mediated tumorigenesis. In situ estrogen production by aromatase is a critical determinant for breast cancer growth and progression. On the contrary, clinical and in vitro studies indicate that androgens have a protective role in mammary carcinogenesis. Here, we demonstrated, in hormone-dependent breast cancer cells, the existence of a functional interplay between the androgen receptor (AR), the orphan nuclear receptor DAX-1 and the aromatase enzyme involved in the inhibition of the estrogen-dependent breast cancer cell proliferation exerted by androgen signaling. Indeed, our results revealed, in MCF-7 cells, that ligand-activated AR induces the expression of the orphan nuclear receptor DAX-1 by direct binding to a newly identified androgen-response-element within the DAX-1 proximal promoter. In turn, androgen-induced DAX-1 is recruited, in association with the corepressor N-CoR, within the SF-1/LRH-1 containing region of the aromatase promoter, thereby repressing aromatase expression and activity. In elucidating a novel mechanism by which androgens, through DAX-1, inhibit aromatase expression in breast cancer cell lines, these findings reinforce the theory of androgen- opposing estrogen-action, opening new avenues for therapeutic intervention in estrogen-dependent breast tumors.

Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression.

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Peroxisome proliferator-activated receptor gamma inhibits follicular and anaplastic thyroid carcinoma cells growth by upregulating p21Cip1/WAF1 gene in a Sp1-dependent manner.

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been demonstrated to be anti-neoplastic against various human tumors. The aim of this study was to delineate the molecular mechanism underlying PPARgamma ligand rosiglitazone (BRL) antiproliferative effects in follicular WRO and anaplastic FRO human thyroid carcinoma cells. BRL upregulated the p21Cip1/WAF1 levels in the two thyroid cancer cells, while did not modify the p53 protein content. Different evidences indicate that the p21Cip1/WAF1 upregulation by BRL requires a functional PPARgamma, since it was reversed by silencing PPARgamma and pretreatment with GW9662, an irreversible PPARgamma antagonist. Transient transfection assays showed that BRL triggered the transcriptional activity of p21Cip1/WAF1 promoter gene in a p53-independent way, being a p21Cip1/WAF1 promoter construct deleted in the p53 sites still activated by BRL. The Sp1 inhibitor mithramycin silenced the p21Cip1/WAF1 promoter activity suggesting an important role of Sp1 in mediating BRL activation. The electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays evidenced a functional interaction between PPARgamma and Sp1 in regulating p21Cip1/WAF1. Intriguingly, ChIP analysis revealed in the p21Cip1/WAF1 gene promoter an increased recruitment of the RNA Pol II associated with an increased histone H3 acetylation and a reduced H3 methylation. The biological event, consistent with PPARgamma-induced WRO and FRO cell growth inhibition, was reversed by p21Cip1/WAF1 antisense oligonucleotides and was confirmed by increasing the PPARgamma expression, suggesting a crucial role exerted by p21Cip1/WAF1 in PPARgamma action. Our results further candidate BRL as a potential agent able to inhibit tumor progression of follicular and anaplastic thyroid carcinoma.

The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells.

Previous epidemiological reports have suggested that red wine intake is associated with beneficial health effects due to the ability of certain phytochemical components to exert estrogen-like activity. It has been also documented that estrogens induce the proliferation of hormone-dependent breast cancer cells by binding to and transactivating estrogen receptor (ER) alpha, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER alpha; (2) they activate the agonist-dependent activation function (AF) 2 of ER alpha and ER beta in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER alpha; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17beta-estradiol for binding to ER alpha and ER beta; and--as a biological counterpart of the aforementioned abilities--(6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.

Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct activation of oestrogen receptor alpha.

Environmental contamination with a variety of industrial products has been associated with developmental and reproductive abnormalities in wildlife species. Increasing evidence has suggested that bisphenol A (BPA) and 4-nonylphenol (NPH), two major endocrine-disrupting chemicals, might be responsible for adverse effects on humans as a consequence of ubiquitous use together with potential oestrogen-like activity. To provide insight into the oestrogen-like nature of BPA and NPH, their ability to activate a reporter gene construct via an oestrogen response element in the hormone-dependent breast cancer cell lines MCF7 and T47D was ascertained. Both compounds transactivated the endogenous oestrogen receptor (ER) alpha in a direct fashion since the anti-oestrogen 4-hydroxytamoxifen abolished the response. In addition, using steroid-receptor-negative HeLa cells engineered to express ERalpha and ERbeta and the hormone-binding domains of both ERalpha and ERbeta, BPA and NPH confirmed the direct transcriptional activity. Interestingly these properties were supported in MCF7 cells by the ability to autoregulate ERalpha expression as well as to induce its nuclear compartmentalization. We therefore evaluated by reverse transcriptase polymerase chain reaction the expression of oestrogen-controlled genes such as cathepsin D and TFF1 (formerly pS2), which were increased by both chemicals tested. The agonistic effects exhibited in all assays performed prompted the evaluation of a more complex biological response such as the proliferation of MCF7 and T47D cells. The same concentration of xenoestrogens eliciting substantial transcriptional activity significantly stimulated the proliferation of both breast cancer cell lines, although with a reduced effectiveness with respect to the natural hormone 17beta-oestradiol. The results indicate that the biological action of environmental oestrogen such as BPA and NPH should be taken into account for the potential impact on human disease-like hormone-dependent breast cancer. However, further studies are needed to clarify their bioavailability and metabolism as well as whether compound mixtures could produce noticeable effects by synergistic activity.

The direct proliferative stimulus of dehydroepiandrosterone on MCF7 breast cancer cells is potentiated by overexpression of aromatase.

In women after menopause aromatization of adrenal androgens represents the main source of estrogens, which may promote the development of hormone-dependent breast tumor. Several studies have attempted to determine the cell type within carcinomas that is responsible for 'in situ' estrogen biosynthesis and whether the amount produced may sustain relevant biological effects. Here we show P450arom mRNA and protein expression together with immunocytochemical localization of aromatase in the epithelial MCF7 breast cancer cell line. Moreover, we demonstrate that the enhanced aromatization of dehydroepiandrosterone in aromatase transfected MCF7 cells confers biological advantages such as proliferative stimulation similar to that induced by estradiol. Our results suggest that aromatase inhibitors may be particularly effective in the treatment of hormone-dependent breast cancer disease in postmenopausal women.

Estrogen receptor alpha mediates the proliferative but not the cytotoxic dose-dependent effects of two major phytoestrogens on human breast cancer cells.

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ER alpha by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ER alpha mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ER alpha and ER beta fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ER alpha and ER beta confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ER alpha-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.

Bone mineral density is inversely related to parathyroid hormone in adolescent girls.

Preventive programs aimed at maximizing peak bone mass as a way of reducing the risk of osteoporotic fractures later in life should take into account the contribution of nutritional factors to bone mass accumulation in young age. The role of calcium and energy intakes on radial mineral density was investigated in 200 healthy girls (aged 11-15 yr) simultaneously evaluating serum changes of insulin-like growth factor-I (IGF-I), parathyroid hormone (PTH) and osteocalcin (OC). Dietary calcium and energy intakes were assessed by a 3-day food record method, bone mineral density (BMD) was performed at ultradistal (ud) and proximal (pr) radial sites using dual energy X-ray absorptiometry. Calcium consumption below the levels suggested by Dietary Reference Intakes in more than 80% of population studied was not related to BMD, which in turn markedly increased in post-compared to premenarcheal girls. Interestingly, in a multiple regression analysis PTH was inversely related to BMD after adjustment for calcium intake, bone age and menarche. Serum IGF-I was positively associated to energy intakes and bone age in girls before menarche, who exhibited the highest values of OC. Our data highlighted the role of food habits in modulating some hormonal response that might influence bone mineral apposition during adolescent age. Low calcium consumption associated to enhanced PTH values, if persisting, could be responsible for reduced rate of gain in bone mineral density. Thus, to optimize bone mineralization during the critical period of rapid body growth adequate intakes of calcium and energy should be recommended.

Parathyroid hormone is elevated but bone markers and density are normal in young female subjects who consume inadequate dietary calcium.

Dietary Ca and osteocalcin (OC), parathyroid hormone (PTH), 25-hydroxyvitamin D (25-OH-D), insulin-like growth factor (IGF)-I and sex hormone binding globulin (SHBG) were assessed simultaneously to bone mineral density (BMD) in 200 adolescent girls (aged 11-15 years) and 100 young women (aged 20-23 years), selected from the lowest and highest end of the Ca intake distribution of a larger population sample. Ca intake was evaluated by food frequency questionnaires, BMD was measured by dual energy x-ray absorptiometry at ultradistal and proximal radius of non-dominant arm, bone age was estimated from x-rays of left hand and wrist according to Tanner et al. (1983). Surprisingly, mean Ca intakes were below the dietary reference intakes in the subgroups of girls and women with the highest measured Ca consumption. Postmenarcheal, but not premenarcheal girls showed radial densities as high as the women and in no group was BMD associated with Ca intake. In all adolescents serum PTH was negatively related to dietary Ca. In girls before menarche IGF-I was positively associated with bone age, while in the same subjects the negative relationship between SHBG and BMD pointed to the crucial role of bioavailable sex steroids on bone mass apposition in early puberty. OC levels decreased progressively with age, while serum 25-OH-D significantly increased after menarche. In conclusion, although in adolescents low Ca intake has not been shown to induce any immediate deleterious effect on radial density, the compensatory hypersecretion of PTH supports the need for an adequate Ca intake to achieve peak bone mass.

Critical years and stages of puberty for radial bone mass apposition during adolescence.

The acquisition of radial mineral density was evaluated in relation to anthropometric characteristics, menarche status, calcium intake and physical activity in a healthy young female population (200 girls and 100 women, respectively aged 11-16 yrs and 20-24 yrs) living in an area of Southern Italy. We performed bone mineral density (BMD) by dual energy X-ray absorptiometry on the ultradistal and middistal radius. Dietary calcium intake was evaluated by a detailed Food Frequency Questionnaire and confirmed by a 3-day record. A questionnaire on energy expenditure was used to assess physical activity in each participant. Morning blood samples were drawn from fasting girls to measure 25-hydroxycalciferol (25 OH-D). We found current calcium above the levels reported by Recommended Dietary Allowances (RDA) in only 31% of women and 6% of girls. BMD steadily increased up to the age of 16 and was increased in postmenarcheal girls compared to premenarcheals of the same pubertal stage. Bone density was also significantly related to age, weight and height in postmenarcheal adolescents, while in girls before and after menarche, no relation was observed between radial BMD and calcium intake or physical activity. In the presence of comparable calcium-intake values recorded in pre- and in postmenarcheal girls, the latter subgroup displayed a marked increase of 25 OH-D serum levels. Our study revealed a calcium intake lower than the RDA in a large percentage of healthy girls and young women, and emphasized the importance of menarche occurrence in bone mass acquisition during pubertal development.

The effect of dietary calcium intake on bone mineral density in healthy adolescent girls and young women in southern Italy.

BACKGROUND: The association between forearm bone mineral areal density (BMD) and dietary calcium, anthropometric characteristics, puberty, and physical activity was studied for the first time in 200 girls (aged 11-15 years) and 100 women (aged 20-23 years) living in Southern Italy. METHODS: The BMD was assessed by dual energy x-ray absorptiometry at ultradistal (ud) and proximal (pr) radial sites and dietary calcium was evaluated using Food Frequency Questionnaires and detailed 3-day food records. RESULTS: For population samples grouped according to low and high calcium intake levels, forearm densities were quite similar among both girls and women. Independently of calcium intake, girls displayed strong correlations between ud/pr-BMD and age, bone age, weight, height and BMI. Furthermore, in girls of similar age and BMI, radial densities were substantially increased following menarche. Positive relationships between weight, BMI and both ud/pr-BMD were only evident in women with high calcium intake. CONCLUSIONS: This study showed that different calcium intake values do not appear to affect forearm mineral densities at the ages investigated, however puberty represents the major event in radial bone mass acquisition during adolescence.

The effect of papaverine on synaptosomal membrane potential.

The authors studied the effect of graded concentrations of papaverine on membrane potential in isolated synaptosomes from pig brain by fluorescence. The indicator dye for membrane potential is rhodamine 6G (Rh6G). The membrane voltage is determined at both high (127.2) and low (30 mM) final sodium (Na+) concentrations. The results demonstrate a hyperpolarizing action of papaverine directly proportional to papaverine concentration.