An Integrated Approach of Bioassays and Non-Target Screening for the Assessment of Endocrine-Disrupting Activities in Tap Water and Identification of Novel Endocrine-Disrupting Chemicals.

The safety of drinking water is a significant environmental issue of great concern for human health since numerous contaminants are often detected in drinking water and its sources. Boiling is a common household method used to produce relatively high-quality drinking water in some countries and regions. In this study, with the aid of an integrated approach of in vitro bioassays and non-target analysis based on high-resolution mass spectrometry coupled with liquid chromatography, alterations in endocrine-disrupting activities in tap water samples without and with boiling were revealed, as well as the potential endocrine-disrupting chemicals (EDCs) contributing to these alterations were identified. The organic extracts of tap water had no significant (ant)agonistic activities against an estrogen receptor (ER), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) at enrichment concentrations of ≤10 times, posing no immediate or acute health risk to humans. However, the presence of agonistic activities against PR and MR and antagonistic activities against ER, PR, GR, and MR in OEs of tap water at relatively higher enrichment concentrations still raise potential health concerns. Boiling effectively reduced antagonistic activities against these steroid hormone receptors (SHRs) but increased estrogenic and glucocorticoid activities in drinking water. Four novel potential EDCs, including one UV filter (phenylbenzimidazole sulfonic acid, PBSA) and three natural metabolites of organisms (beta-hydroxymyristic acid, 12-hydroxyoctadecanoic acid, and isorosmanol) were identified in drinking water samples, each of which showed (ant)agonistic activities against different SHRs. Given the widespread use of UV filters in sunscreens to prevent skin cancer, the health risks posed by PBSA as an identified novel EDC are of concern. Although boiling has been thought to reduce the health risk of drinking water contamination, our findings suggest that boiling may have a more complex effect on the endocrine-disrupting activities of drinking water and, therefore, a more comprehensive assessment is needed.

Phytosterols activating nuclear receptors are involving in steroid hormone-dependent cancers: Myth or fact?

Nuclear receptors (NRs) represent intracellular proteins that function as a signaling network of transcriptional factors to control genes in response to a variety of environmental, dietary, and hormonal stimulations or serve as orphan receptors lacking a recognized ligand. They also play an essential role in normal development, metabolism, cell growth, cell division, physiology, reproduction, and homeostasis and function as biological markers for tumor subclassification and as targets for hormone therapy. NRs, including steroid hormone receptors (SHRs), have been studied as tools to examine the fundamentals of transcriptional regulation within the development of mammals and human physiology, in addition to their links to disturbances. In this regard, it is widely recognized that aberrant NR signaling is responsible for the pathological growth of hormone-dependent tumors in response to SHRs dysregulation and consequently represents a potential therapeutic candidate in a range of diseases, as in the case of prostate cancer and breast cancer. On the other hand, phytosterols are a group of plant-derived compounds that act directly as ligands for NRs and have proven their efficacy in the management of diabetes, heart diseases, and cancers. However, these plants are not suggested in cases of hormone-dependent cancer since a certain group of plants contains molecules with a chemical structure similar to that of estrogens, which are known as phytoestrogens or estrogen-like compounds, such as lignans, coumestans, and isoflavones. Therefore, it remains an open and controversial debate regarding whether consuming a phytosterol-rich diet and adopting a vegetarian lifestyle like the Mediterranean diet may increase the risk of developing steroid hormone-dependent cancers by constitutively activating SHRs and thereby leading to tumor transformation. Overall, the purpose of this review is to better understand the relevant mechanistic pathways and explore epidemiological investigations in order to establish that phytosterols may contribute to the activation of NRs as cancer drivers in hormone-dependent cancers.

Structure and functions of the N-terminal domain of steroid hormone receptors.

The steroid hormone receptors (SHRs) belong to the large superfamily of nuclear receptors that selectively modulate gene expression in response to specific hormone ligands. The SHRs are required in a broad range of normal physiological processes as well as associated with numerous pathological conditions. Over years, the understanding of the SHR biology and mechanisms of their actions on target cells have found many clinical applications and management of various endocrine-related disorders. However, the effectiveness of SHR-based therapies in endocrine-related cancers remain a clinical challenge. This, in part, is due to the lack of in-depth understanding of structural dynamics and functions of SHRs' intrinsically disordered N-terminal domain (NTD). Recent progress in delineating SHR structural information and their correlations with receptor action in a highly dynamic environment is ultimately helping to explain how diverse SHR signaling mechanisms can elicit selective biological effects. Recent developments are providing new insights of how NTD's structural flexibility plays an important role in SHRs' allosteric regulation leading to the fine tuning of target gene expression to more precisely control SHRs' cell/tissue-specific functions. In this review article, we are discussing the up-to-date knowledge about the SHR actions with a particular emphasis on the structure and functions of the NTD.

Maternal exposure to di(2-ethylhexyl) phthalate (DEHP) causes multigenerational adverse effects on the uterus of F1 and F2 offspring rats.

Phthalates are one of the ubiquitous chemicals found in day-to-day products like food packaging, children's toys, and other consumer commodities. There is rising concern that repeated exposure to phthalates during pregnancy and lactation could have long-term effects on maternal and fetal health. We hypothesize that exposure to DEHP during the developmental windows might affect the expression of molecules that regulate uterine function and that this effect would be passed on to further generations. Rat dams were treated with olive oil (vehicle) or DEHP (100 mg/kg b.wt./day) orally from gestational day 9 (GD 9) to the end of lactation (PND 21). F0 maternal DEHP exposure resulted in multigenerational (F1 and F2) reproductive toxicity, as evidenced by an extended estrous cycle, decreased mating, fertility, and fecundity indices. Serum progesterone and estradiol levels were decreased and their cognate receptors (PR and ERα) in the uterus were decreased in the DEHP-exposed offspring rats. Further analysis of the expression of estrogen and progesterone regulatory genes such as Hox a11, VEGF A, Ihh, LIFR, EP4, PTCH, NR2F2, BMP2, and Wnt4 were reduced in the uteri of adult F1 and F2 generation rats born from DEHP-exposed F0 dams. Decreased expression of these crucial proteins due to DEHP exposure may lead to defects in epithelial proliferation and secretion, uterine receptivity, and decidualization in the uteri of successive generations. This study showed that maternal DEHP exposure impairs the expression of molecules that regulate uterine function and this multigenerational effect is transmitted via maternal lineage.

Effect of Sex Steroids and PGF2α on the Expression of Their Receptors and Decorin in Bovine Caruncular Epithelial Cells in Early-Mid Pregnancy.

Changes in the expression of various genes, including pregnancy-associated hormone receptors and extracellular matrix proteins, have been suggested to play a significant role in bovine placental development. This study aimed to examine the influence of sex steroids and PGF2α on decorin (DCN) expression in the epithelial cells of bovine caruncle in early−mid pregnancy in cows. The expression patterns of DCN, PTGFR, PGR and ESR1 were analyzed by RT-qPCR and Western blotting in primary caruncular epithelial cell cultures (PCECC) and placental tissue homogenates derived from the 2nd and 4th months of pregnancy. PCECC were found to express DCN, PTGFR, PGR and ESR1. The intensity of PGR staining was higher in cells derived from the 4th month of pregnancy (p < 0.05). The 17ß-estradiol, progesterone and PGF2α have not been shown to affect DCN expression. PGF2α decreased PTGFR expression in cells derived from the 4th month of gestation (p < 0.05). In conclusion, the results of the present preliminary study showed that the expression of the PTGFR, ESR1, PGR and DCN in PCECC does not vary throughout early−mid pregnancy. Further studies should be carried out to observe the relationship between hormonal status and cellular adhesion to determine their importance for properly developing placentation and pregnancy in cows.

A specialized Hsp90 co-chaperone network regulates steroid hormone receptor response to ligand.

Heat shock protein-90 (Hsp90) chaperone machinery is involved in the stability and activity of its client proteins. The chaperone function of Hsp90 is regulated by co-chaperones and post-translational modifications. Although structural evidence exists for Hsp90 interaction with clients, our understanding of the impact of Hsp90 chaperone function toward client activity in cells remains elusive. Here, we dissect the impact of recently identified higher eukaryotic co-chaperones, FNIP1/2 (FNIPs) and Tsc1, toward Hsp90 client activity. Our data show that Tsc1 and FNIP2 form mutually exclusive complexes with FNIP1, and that unlike Tsc1, FNIP1/2 interact with the catalytic residue of Hsp90. Functionally, these co-chaperone complexes increase the affinity of the steroid hormone receptors glucocorticoid receptor and estrogen receptor to their ligands in vivo. We provide a model for the responsiveness of the steroid hormone receptor activation upon ligand binding as a consequence of their association with specific Hsp90:co-chaperone subpopulations.

Targeting steroid hormone receptors for anti-cancer therapy-A review on small molecules and nanotherapeutic approaches.

The steroid hormone receptors (SHRs) among nuclear hormone receptors (NHRs) are steroid ligand-dependent transcription factors that play important roles in the regulation of transcription of genes promoted via hormone responsive elements in our genome. Aberrant expression patterns and context-specific regulation of these receptors in cancer, have been routinely reported by multiple research groups. These gave an window of opportunity to target those receptors in the context of developing novel, targeted anticancer therapeutics. Besides the development of a plethora of SHR-targeting synthetic ligands and the availability of their natural, hormonal ligands, development of many SHR-targeted, anticancer nano-delivery systems and theranostics, especially based on small molecules, have been reported. It is intriguing to realize that these cytoplasmic receptors have become a hot target for cancer selective delivery. This is in spite of the fact that these receptors do not fall in the category of conventional, targetable cell surface bound or transmembrane receptors that enjoy over-expression status. Glucocorticoid receptor (GR) is one such exciting SHR that in spite of it being expressed ubiquitously in all cells, we discovered it to behave differently in cancer cells, thus making it a truly druggable target for treating cancer. This review selectively accumulates the knowledge generated in the field of SHR-targeting as a major focus for cancer treatment with various anticancer small molecules and nanotherapeutics on progesterone receptor, mineralocorticoid receptor, and androgen receptor while selectively emphasizing on GR and estrogen receptor. This review also briefly highlights lipid-modification strategy to convert ligands into SHR-targeted cancer nanotherapeutics. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Biology-Inspired Nanomaterials > Lipid-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Nonclassical androgen and estrogen signaling is essential for normal spermatogenesis.

Signaling by androgens through androgen receptor (AR) is essential to complete spermatogenesis in the testis. Similarly, loss of the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), results in male infertility, due in part to indirect deleterious effects on the seminiferous epithelium and spermatogenesis. Effects of steroid hormones are induced primarily through genomic changes induced by hormone-mediated activation of their intracellular receptors and subsequent effects on nuclear gene transcription. However, androgens and estrogens also signal through rapid nonclassical pathways involving actions initiated at the cell membrane. Here we review the data that nonclassical androgen and estrogen signaling pathways support processes essential for male fertility in the testis and reproductive tract. The recent development of transgenic mice lacking nonclassical AR or ESR1 signaling but retaining genomic nuclear signaling has provided a powerful tool to elucidate the function of nonclassical signaling in the overall response to androgens and estrogens. Results from these mice have emphasized that nonclassical signaling is essential for full responses to these hormones, and absence of either nonclassical or classical AR or ESR1 pathways produces abnormalities in spermatogenesis and the male reproductive tract. Although additional work is required to fully understand how classical and nonclassical receptor signaling synergize to produce full steroid hormone responses, here we summarize the known physiological functions of the classical and nonclassical androgen and estrogen signaling pathways in the testis and reproductive tract.

Sex-specific effects of 17ß-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERß and require palmitoylation to translocate the receptors to the plasma membrane.

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17ß-estradiol (E2), testosterone, and dihydrotestosterone (DHT). Mechanistically, E2 and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERß) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E2 signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERß and required intact caveolae; PKC activity was inhibited by the E2 enantiomer and by an inhibitor of ERß. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E2 treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERß also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E2 and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERß is required for E2 and DHT membrane-associated PKC activity in costochondral cartilage cells.

Steroid Hormone Receptors: Links With Cell Cycle Machinery and Breast Cancer Progression.

Progression of cells through cell cycle consists of a series of events orchestrated in a regulated fashion. Such processes are influenced by cell cycle regulated expression of various proteins where multiple families of transcription factors take integral parts. Among these, the steroid hormone receptors (SHRs) represent a connection between the external hormone milieu and genes that control cellular proliferation. Therefore, understanding the molecular connection between the transcriptional role of steroid hormone receptors and cell cycle deserves importance in dissecting cellular proliferation in normal as well as malignant conditions. Deregulation of cell cycle promotes malignancies of various origins, including breast cancer. Indeed, SHR members play crucial role in breast cancer progression as well as management. This review focuses on SHR-driven cell cycle regulation and moving forward, attempts to discuss the role of SHR-driven crosstalk between cell cycle anomalies and breast cancer.

The fungicide Mancozeb reduces spheroid attachment onto endometrial epithelial cells through downregulation of estrogen receptor ß and integrin ß3 in Ishikawa cells.

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 µg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 µg/mL and ETU at 300 µg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 µg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor ß and integrin ß3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor ß and integrin ß3 expression of endometrial epithelial cells.

Investigating the NPY/AgRP/GABA to GnRH Neuron Circuit in Prenatally Androgenized PCOS-Like Mice.

Polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility, is associated with altered signaling within the hormone-sensitive neuronal network that regulates gonadotropin-releasing hormone (GnRH) neurons, leading to a pathological increase in GnRH secretion. Circuit remodeling is evident between GABAergic neurons in the arcuate nucleus (ARN) and GnRH neurons in a murine model of PCOS. One-third of ARN GABA neurons co-express neuropeptide Y (NPY), which has a known yet complex role in regulating GnRH neurons and reproductive function. Here, we investigated whether the NPY-expressing subpopulation (NPYARN) of ARN GABA neurons (GABAARN) is also affected in prenatally androgenized (PNA) PCOS-like NPYARN reporter mice [Agouti-related protein (AgRP)-Cre;τGFP]. PCOS-like mice and controls were generated by exposure to di-hydrotestosterone or vehicle (VEH) in late gestation. τGFP-expressing NPYARN neuron fiber appositions with GnRH neurons and gonadal steroid hormone receptor expression in τGFP-expressing NPYARN neurons were assessed using confocal microscopy. Although GnRH neurons received abundant close contacts from τGFP-expressing NPYARN neuron fibers, the number and density of putative inputs was not affected by prenatal androgen excess. NPYARN neurons did not co-express progesterone receptor or estrogen receptor α in either PNA or VEH mice. However, the proportion of NPYARN neurons co-expressing the androgen receptor was significantly elevated in PNA mice. Therefore, NPYARN neurons are not remodeled by prenatal androgen excess like the wider GABAARN population, indicating GABA-to-GnRH neuron circuit remodeling occurs in a presently unidentified non-NPY/AgRP population of GABAARN neurons. NPYARN neurons do, however, show independent changes in the form of elevated androgen sensitivity.

Endometrial changes in estrogen and progesterone receptor expression during implantation in an oocyte donation program.

Implantation is the final and most important stage of embryogenesis and is of paramount importance in achieving a successful pregnancy. Progesterone and estrogen are steroid hormones responsible for the regulation of the implantation window and the current study hypothesised that their receptors may be implicated in women undergoing oocyte donation. A total of 15 women aged 25-32 years old (mean ± SD, 28.9±2.89) undergoing oocyte donation were recruited into the present study. Participants underwent ovarian stimulation with gonadotrophin-releasing hormone antagonist and recombinant follicle-stimulating hormone. Endometrial aspiration biopsy was performed on the day of oocyte retrieval and after 5 days (on days 0 and 5, respectively). Endometrial histology and evaluation of estrogen receptor (ER)α and progesterone receptor (PR)-B were performed on days 0 and 5. The ER nodal staining percentage on day 0 was age-associated, with patients aged <30 years demonstrating 100% staining and those aged >30 years exhibiting 90% staining. Pathological staining revealed statistically significant differences between days 0 and 5 following all staining procedures. Wilcoxon signed-rank test resulted in the following P-values, for ER (nodes % and stromal %) day 0/5, P=0.0001; for PR (nodes % and stromal %) day 0/5, P=0.0001 and P=0.035, respectively; for ER (grade nodes and stromal %) day 0/5, P=0.0001; and PR (grade nodes and stromal %) day 0/5 P=0.0001 and P=0.016, respectively. Synchronization between blastocyst development and the acquisition of endometrial receptivity is a prerequisite for the success of in vitro fertilisation (IVF). Aside from the recent discovery of molecules that are considered crucial for successful embryo implantation, assessing the functional characteristics of the endometrium may offer unique insights into this process, thus improving IVF results.

Nuclear receptor phosphorylation in xenobiotic signal transduction.

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

Quantitative Evaluation of the Transcriptional Activity of Steroid Hormone Receptor Mutants and Variants Using a Single Vector With Two Reporters and a Receptor Expression Cassette.

Although the rapid development of high-throughput sequencing has led to the identification of a large number of truncated or mutated steroid hormone receptor (SHR) variants, their clinical relevance remains to be defined. A platform for functional analysis of these SHR variants in cells would be instrumental for better assessing their impact on normal physiology and SHR-associated diseases. Here we have developed a new reporter system that allows rapid and accurate assessment of the transcriptional activity of SHR variants in cells. The reporter is a single construct containing a firefly luciferase reporter gene, whose expression is under the control of a promoter with multiple steroid hormone responsive elements, and a Renilla luciferase reporter gene, that is constitutively expressed under the control of an internal ribosome entry site (IRES) and is not regulated by steroid hormones. The corresponding SHR (wildtype or mutant/variant) is also expressed from the same construct. Using this improved reporter system, we revealed a large spectrum of transactivation activities within a set of previously identified mutations and variations of the androgen receptor (AR), the estrogen receptor α (ERα) and the glucocorticoid receptor (GR). This novel reporter system enables functional analysis of SHR mutants and variants in physiological and pathological settings, offering valuable preclinical, or diagnostic information for the understanding and treatment of associated diseases.

Spatial and temporal changes in the expression of steroid hormone receptors in mouse model of endometriosis.

PURPOSE: Endometriosis is recognized as a steroid hormone-dependent disorder. However, controversies exist regarding the status of the steroid hormone receptor expression in endometriotic tissues. The purpose of this study was to determine the ontogeny of cellular changes in the expression of estrogen receptors (ERα, ERß), G protein-coupled estrogen receptor 1 (GPER1), and progesterone receptors (PRs) in endometriosis using a mouse model. METHODS: We used the autologous uterine tissue transfer mouse model and studied the mRNA and protein expression of ERα, ERß, GPER1, and PR in ectopic lesions at 2, 4, and 8 weeks of induction of endometriosis. RESULT: As compared to endometrium of controls, in the ectopic endometrium, ERα is reduced while ERß was elevated in stromal cells; however, Gper1 and PR levels are reduced in both stromal and epithelial cells in a time-specific manner. There is a high inter-animal variation in the levels of these receptors in ectopic endometrium as compared to controls; the levels also varied by almost 100-fold within the same lesion resulting in "micro-heterogeneity." The expression of all these receptors also deferred between two lesions from the same animal. CONCLUSION: In the endometriotic tissue, there is extensive inter-animal and intra-lesion heterogeneity in the expression of ERα, ERß, GPER1, and PR. These changes are not due to the influence of the peritoneal environment but appear to be tissue intrinsic. We propose that the variable outcomes in hormonal therapy for endometriosis could be possibly due to heterogeneity in the expression of steroid hormone receptors in the ectopic endometrium.

Chronic and Low Dose Exposure to Nonlyphenol or Di(2-Ethylhexyl) Phthalate Alters Cell Proliferation and the Localization of Steroid Hormone Receptors in Uterine Endometria in Mice.

Based on our preliminary results, we examined the possible role of low-dose and chronic-exposing of the chemicals those are known as endocrine disrupting chemical (EDC), on the proliferation of uterine endometrium and the localization of steroid receptors. Immunohistochemical or immunofluorochemical methodology were employed to evaluate the localization of antigen identified by monoclonal antibody Ki 67 protein (MKI67), estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), and progesterone receptor (PGR). In 133 µg/L and 1,330 µg/L di(2-ethylhexyl) phthalate (DEHP) and 50 µg/L nonylphenol (NP) groups, the ratio of MKI67 positive stromal cells was significantly increased but not in 500 µg/L NP group. The ratios of MKI67 positive glandular and luminal epithelial cells were also changed by the chronic administration of NP and DEHP in tissue with dose specific manner. ESR1 signals were localized in nucleus in glandular and luminal epithelia of control group but its localization was mainly in cytoplasm in DEHP and NP administered groups. On the other hand, it was decreased at nucleus of stromal cells in 1,330 µg/L DEHP group. The colocalization patterns of these nuclear receptors were also modified by the administration of these chemicals. Such a tissue specific and dose specific localization of ESR2 and PGR were detected as ESR1 in all the uterine endometrial tissues. These results show that the chronic lows-dose exposing of NP or DEHP modify the localization and colocalization of ESRs and PGR, and of the proliferation patterns of the endometrial tissues.

Forkhead box O1 as an indicator of prognosis is inactivated in urothelial carcinoma of the upper urinary tract.

The transcription factor forkhead box O1 (FOXO1) can be inactivated via its phosphorylation, resulting in suppression of apoptosis. Using immunohistochemistry, the expression of a phosphorylated form of FOXO1 was assessed in upper urinary tract urothelial carcinoma (UUTUC) specimens. Overall, phospho-FOXO1 (p-FOXO1) was immunoreactive in all 99 UUTUC specimens [12 (12.1%) weak (1+), 46 (46.5%) moderate (2+) and 41 (41.4%) strong (3+)], which was significantly (P=0.018) increased, compared with benign urothelium specimens [77/82 (93.9%): 18 (22.0%) 1+, 41 (50.0%) 2+ and 18 (22.0%) 3+]. Muscle invasion (P=0.031) and lymphovascular invasion (P=0.025) were observed more frequently in p-FOXO1(2+/3+) tumor samples compared with p-FOXO1(1+) tumor samples. No statistically significant associations between p-FOXO1 expression and tumor grade or presence of concurrent carcinoma in situ, hydronephrosis or lymph node metastasis were observed. Furthermore, the levels of p-FOXO1 and estrogen receptor-ß expression were significantly (P<0.05) correlated in UUTUC samples [correlation coefficient (CC)=0.244], particularly in tumor samples from male patients (CC=0.330). Additionally, patients with p-FOXO1(3+) tumors had a significantly increased risk of cancer-specific mortality (P=0.043), compared with those with p-FOXO1(1+/2+) tumors. Multivariate analysis further demonstrated a notable, albeit not significant, association between p-FOXO1 expression and cancer-specific survival (hazard ratio=2.204; P=0.053). These findings indicate that FOXO1 is inactivated in UUTUC specimens and p-FOXO1 overexpression may serve as a predictor of poor patient outcomes.

Estradiol receptor profile and estrogen responsiveness in laryngeal cancer and clinical outcomes.

There is growing evidence that laryngeal cancers are responsive to sex hormones, specifically 17ß-estradiol (E2), despite controversy regarding the presence and characterization of E2 receptors (ER). Determination of sex hormone responsiveness impacts the prognosis of laryngeal cancer patients and the treatment modalities implemented by their clinicians. Discovery of membrane-associated steroid hormone receptors and rapid membrane signaling opened the possibility that cancers previously labeled 'non-hormone dependent' and 'ER negative' might in fact be susceptible to the effects of E2 via these membrane receptors. ERα66 and ERß, the classical nuclear receptors, are present in the membranes of different cancer cells via a mechanism referred to as trafficking. Novel splice variants of these traditional receptors, a key example being ERα36, have also been found in the caveolae of cancer cells. Previous work demonstrated that ERα36 has a role in the tumorigenesis of laryngeal cancer, enhancing both proliferation and the anti-apoptotic effect of E2 against chemotherapeutics. The present study showed that expression of different membrane ERs in laryngeal cancer is not uniform, which may result in differential and even antagonistic responses to E2. E2 had protective or deleterious effects in different cancer cell lines, stimulating proliferation and conferring anti-apoptotic potential to the cancer cells according to their receptor profile. These findings stress the importance of establishing the molecular and clinical characterization of the specific laryngeal tumor in order to tailor treatment accordingly, thus optimizing care while reducing adverse effects for individual patients.

Molecular cloning of androgen receptor and gene expression of sex steroid hormone receptors in the brain of newborn Chinese alligator (Alligator sinensis).

Sex steroid hormones play an important role in mediating physiological responses and developmental processes through their receptors across all vertebrates. Chinese alligator (Alligator sinensis) is a critically endangered reptile species unique to China. In this study, we have cloned one of the sex steroid hormone receptor genes, androgen receptor (AR) from the brain of Chinese alligator for the first time. The full-length AR cDNA is 2717 bp in length with an open reading frame (ORF) encoding 722 amino acids. Amino acid alignment analyses indicated that the ARs exhibit highly conserved functional domains. Especially, the P-box and D-box, which are essential to ensure that receptor binding to the androgen response elements, are completely conserved in selected species. Using the quantitative real-time PCR (qPCR), the spatial expression of four receptor mRNAs in all newborn brain tissues and temporal expression of them in the cerebrum during the embryonic development in Chinese alligators were investigated. The results of qPCR showed ubiquitous expression of the four receptor mRNAs in all newborn brain tissues examined and significant changes in the expression levels of these receptor mRNAs in the embryonic development. These results suggest that sex steroid hormones might play an important role in the regulation of complex neuroendocrine activities in newborn Chinese alligator. Furthermore, these data provide an important foundation for further studies on endocrinology and molecular biology of non-mammalian sex steroid hormone receptors.