Estrogen, Progesterone and Epithelial Ovarian Cancer

Ovarian carcinoma (OCa) continues to be the leading cause of death due to gynecologic malignancies and the vast majority of OCa is derived from the ovarian surface epithelium (OSE) and its cystic derivatives. Epidemiological evidence strongly suggests that steroid hormones, primarily estrogens and progesterone, are implicated in ovarian carcinogenesis. However, it has proved difficult to fully understand their mechanisms of action on the tumorigenic process. New convincing data have indicated that estrogens favor neoplastic transformation of the OSE while progesterone offers protection against OCa development. Specifically, estrogens, particularly those present in ovulatory follicles, are both genotoxic and mitogenic to OSE cells. In contrast, pregnancy-equivalent levels progesterone are highly effective as apoptosis inducers for OSE and OCa cells. In this regard, high-dose progestin may exert an exfoliation effect and rid an aged OSE of pre-malignant cells. A limited number of clinical studies has demonstrated efficacies of antiestrogens, aromatase inhibitors, and progestins alone or in combination with chemotherapeutic drugs in the treatment of OCa. As a result of increased life expectancy in most countries, the number of women taking hormone replacement therapies (HRT) continues to grow. Thus, knowledge of the mechanism of action of steroid hormones on the OSE and OCa is of paramount significance to HRT risk assessment and to the development of novel therapies for the prevention and treatment of OCa

Environmental Epigenetics and Asthma: Current Concepts and Call for Studies

Recent studies suggest that epigenetic regulation (heritable changes in gene expression that occur in the absence of alterations in DNA sequences) may in part mediate the complex gene-by-environment interactions that can lead to asthma. The variable natural history of asthma (i.e., incidence and remission of symptoms) may be a result of epigenetic changes, such as DNA methylation, covalent histone modifications, microRNA changes, and chromatin alterations, after early or later environmental exposures. Findings from multiple epidemiologic and experimental studies indicate that asthma risk may be modified by epigenetic regulation. One study suggested that the transmission of asthma risk may occur across multiple generations. Experimental studies provide substantial in vitro data indicating that DNA methylation of genes critical to T-helper cell differentiation may induce polarization toward or away from an allergic phenotype. Despite this initial progress, fundamental questions remain that need to be addressed by well-designed research studies. Data generated from controlled experiments using in vivo models and/or clinical specimens collected after environmental exposure monitoring are limited. Importantly, cohort-driven epigenetic research has the potential to address key questions, such as those concerning the influence of timing of exposure, dose of exposure, diet, and ethnicity on susceptibility to asthma development. There is immense promise that the study of environmental epigenetics will help us understand a theoretically preventable environmental disease

Association between plasma fluorescent oxidation products and erectile dysfunction: A prospective study

Background: Existing epidemiological studies of the association between oxidative stress and erectile dysfunction (ED) are sparse and inconclusive, which is likely due to cross-sectional design and small sample size. Therefore, we investigated the association between biomarkers of oxidative stress and ED in prospective setting among a relatively large sample size of men. Methods: We conducted the prospective study among 917 men ages between 47 and 80 years at the time of blood draw, which is a part of nested prospective case–control study of prostate cancer in the Health Professionals Follow-up Study. Plasma fluorescent oxidation products (FlOPs), a global biomarker for oxidative stress, were measured at three excitation/emission wavelengths (360/420 nm named as FlOP_360; 320/420 nm named as FlOP_320 and 400/475 nm named as FlOP_400). Results: Approximately 35 % of men developed ED during follow-up. We did not find an independent association between FlOP_360, FlOP_320, FlOP_400 and risk of ED in the multivariable adjusted model (Tertile 3 vs. tertile 1: odds ratio [OR] = 0.90, 95 % confidence interval [CI] = 0.61-1.34, Ptrend = 0.54 for FlOP_360; OR = 0.73, 95 % CI = 0.49-1.07, Ptrend = 0.27 for FlOP_320; and OR = 0.98, 95 % CI = 0.66-1.45, Ptrend = 0.72 for FlOP_400). Further analysis of the association between FlOPs and ED in the fasting samples or controls only (free of prostate cancer incidence) did not change the results appreciably. Conclusions: Plasma FlOPs were not associated with the risk of ED, suggesting oxidative stress may not be an independent risk factor for ED. Electronic supplementary material The online version of this article (doi:10.1186/s12894-015-0083-9) contains supplementary material, which is available to authorized users

Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation

The aim of the current study is to demonstrate normal and malignant prostatic epithelial cells (PrECs) as targets for receptor-mediated estrogenic and antiestrogenic action. Using an improved protocol, we have successfully isolated and maintained highly enriched populations of normal PrECs from ultrasound-guided peripheral zone biopsies, individually determined to be morphologically normal. Semiquantitative reverse transcription-PCR analyses were used to determine whether transcripts of estrogen receptor (ER)-alpha and those of ER-beta were expressed in our normal PrEC primary cultures, in a commercially available PrEC preparation (PrEC; Clontech), in an immortalized PrEC line established from a benign prostatic hyperplasia specimen (BPH-1), and in three prostatic cancer cell lines (LNCaP, PC-3, and DU145). Expression levels of ER-alpha and ER-beta transcripts were related to those of two estrogen-responsive genes [progesterone receptor (PR) and pS2], at the message levels, to gain insights into the functionality of the ER subtypes in PrECs. Interestingly, only transcripts of ER-beta, but not those of ER-alpha, were found in our primary cultures of normal PrECs, along with both PR and pS2 mRNA. These data strongly suggest that estrogen action was signaled exclusively via ER-beta in normal human PrECs. In contrast, PrEC (Clontech) and BPH-1 cells expressed both ER-alpha and ER-beta transcripts and no PR nor pS2 mRNA in PrEC and only a minimal level of PR mRNA in BPH-1. Among the three prostate cancer cell lines, LNCaP expressed ER-beta mRNA along with transcripts of PR and pS2, DU145 expressed messages of ER-beta and PR, and PC-3 cells exhibited ER-alpha, ER-beta, and pS2 mRNA. Thus, unlike normal PrECs, expression patterns of these genes in malignant PrECs are more variable. Treatment of prostate cancer cells with demethylation agents effectively reactivated the expression of ER-alpha mRNA in LNCaP and DU145 and that of pS2 message in DU145. These findings provide experimental evidence that ER-alpha gene silencing in prostate cancer cells, and perhaps also in normal PrECs, are caused by DNA hypermethylation. To evaluate the potential of using antiestrogens as prostate cancer therapies, we have assessed the growth-inhibitory action of estrogens (estradiol and diethylstilbestrol) and antiestrogens (4-hydroxy-tamoxifen and ICI-182,780) on PC-3 and DU-145 cells. In PC-3 cells, which express both ER subtypes, estrogens as well as antiestrogens are effective inhibitors. In contrast, in DU145 cells, which express only ER-beta, antiestrogens, but not estrogens, are growth inhibitors. By comparison, ICI 182,780 is the more effective cell growth inhibitor. Importantly, the ICI 182,780-induced antiproliferative effects were reversed by cotreatment of DU145 cells with an ER-beta antisense oligonucleotide, hence lending additional support to a central role played by ER-beta in mediating growth-inhibitory action of antiestrogens

Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells

Epidemiological data have implicated reproductive hormones as probable risk factors for ovarian cancer (OCa) development. Although pituitary and sex hormones have been reported to regulate OCa cell growth, no information is available regarding whether and how they influence normal ovarian surface epithelial (OSE) cell proliferation. To fill this data gap, this study has compared cell growth responses to gonadotropins and sex steroids in primary cultures of human OSE (HOSE) cells with those observed in immortalized, nontumorigenic HOSE cells and in OCa cell lines. Both malignant and normal cell lines/cultures responded equally well to the stimulatory actions of luteinizing hormone and follicle-stimulating hormone and to 17beta-estradiol and estrone, although the latter estrogen has a much lower affinity for estrogen receptor than does the former estrogen. In normal HOSE cell cultures/lines, 5alpha-dihydrotestosterone was found to be more effective than testosterone in stimulating cell growth, but in OCa cell lines, 5alpha-dihydrotestosterone and testosterone are equally potent. One OCa cell line, OVCA 433, was found to be nonresponsive to androgen stimulation. In general, primary cultures of normal HOSE cells exhibited the greatest hormone-stimulated growth responses (\u3e10-fold enhancement), followed by immortalized HOSE cell lines (4-5-fold enhancement) and by OCa cell lines (2-4-fold enhancement). Interestingly, progesterone (P4), at low concentrations (10(-11) to 10(-10) M), was stimulatory to HOSE and OCa cell growth, but at high doses (10(-8) to 10(-6) M), P4 exerted marked inhibitory effects. In all cases, cotreatment of a cell culture/line with a hormone and its specific antagonist blocked the effect of the hormone, confirming specificity of the hormonal action. Taken together, these data support the hypothesis that reproductive states associated with rising levels of gonadotropins, estrogen, and/or androgen promote cell proliferation in the normal OSE, which favors neoplastic transformation. Conversely, those states attended by high levels of circulating P4, such as that seen during pregnancy, induce OSE cell loss and offer protection against ovarian carcinogenesis

Overexpression of cytochrome P450 1A1 and its novel spliced variant in ovarian cancer cells: alternative subcellular enzyme compartmentation may contribute to carcinogenesis

Epithelial ovarian cancer derived from the human ovarian surface epithelium (HOSE) is the leading cause of death from gynecologic malignancies among American women. Metabolic activation of endogenous and exogenous chemicals by cytochrome P450 (CYP) class I enzymes has been implicated in its etiology. In this study, we showed overexpression of CYP1A1 mRNA, but not CYP1B1 transcripts, in ovarian cancer cell lines when compared with primary cultures or immortalized HOSE cell lines. Importantly, we identified a novel, enzymatically active, spliced variant of CYP1A1 (CYP1A1v) formed by excision of an 84-bp cryptic intron in exon 2. CYP1A1v is overexpressed in ovarian cancer cell lines and exhibits a unique subcellular distribution restricted to the nucleus and mitochondria, contrary to the endoplasmic reticulum localization of the wild-type enzyme. In concordance, total CYP1A1 activity, as measured by the ethoxyresorufin O-deethylase assay, was detected in mitochondrial, nuclear, and microsomal fractions of ovarian cancer cells but was notably absent in all subcellular fractions of HOSE cells. Immunocytochemistry studies in 30 clinical specimens revealed overexpression of CYP1A1 in various types of ovarian cancers compared with benign epithelia and frequent localization of the enzyme to cancer cell nuclei. Forced expression of CYP1A1wt or CYP1A1v in HOSE cells resulted in nuclear localization of the enzyme and acquisition of anchorage-independent growth, which was further exacerbated following exposure to benzo(a)pyrene or 17beta-estradiol. Collectively, these data provided the first evidence that CYP1A1 overexpression and alternative splicing could contribute to ovarian cancer initiation and progression

Novel Magnetic Resonance Imaging strategy targeting Neurotensin Receptors in detection of Prostate Cancer [preprint]

Prostate cancer is the second leading cause of all male cancer deaths. One of the factors present in malignant prostate cells and shown to support its metastatic growth is the neuropeptide neurotensin (NT). The primary goal of the present study was to establish the feasibility of using a newly developed paramagnetic receptor ligand for NT and non-invasive ultrahigh-field magnetic resonance (MR) imaging to visualize prostate cancer in rodents. Orthotropic xenografts were initiated in six-week old male BALB/c nu/nu athymic mice (n = 28) by intra-prostatic (ventral lobe) inoculation of human prostate cancer cells (10 μL of PC3 cells (10^6/100 μL)). Palpable tumors developed within 30-60 days. A micro-imager utilized in these studies was an actively shielded 9.4T, 89 mm bore, Oxford superconducting magnet with a 100 gauss/cm gradient system. Prior to contrast injection, T2 weighted anatomy scans were done to localize the tumor with a spin-echo multi-slice sequence with TR: 2000 TE: 40 and NEX: 1 in both coronal and axial planes. The paramagnetic ligand data sets were collected with a spin-echo, T1 weighted pulse sequence (MSME): TR 300 msec; TE 5 msec; NEX 4 in both axial and coronal planes. The data sets were taken initially at 5-min intervals post contrast injection for the first half hour and then at 15 min intervals for the next 1.5-2 hours for a time series analyses. The temporal distribution of MR signal intensity in various regions were determined in the absence and presence of NT. Our results confirm that the novel NT molecule was protected from enzymatic degradation and capable of forming a high-affinity paramagnetic NT ligand with an extended half-life. During the imaging studies, the signal intensity increased by 200% in the region of the tumor. This increase in signal intensity approached maximum binding within 30 minutes and remained visible for 1-hour post-injection of the contrast agent. Taken together, these findings suggest that it is feasible to detect and image prostate cancer using a paramagnetic NT ligand and the emergence of the NT receptor ligand that may be used as a diagnostic marker for prostate cancer in humans

Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells

Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC.We treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin beta4, alpha-catenin, karyopherin [importin] beta1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets.We here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells

Estrogen receptor β2 and β5 are associated with poor prognosis in prostate cancer, and promote cancer cell migration and invasion

Estrogens play a pivotal role in the development and progression of prostate cancer (PCa). Their actions are mediated by estrogen receptors (ERs), particularly ERβ in the prostate epithelium. With the discovery of ERβ isoforms, data from previous studies that focused principally on the wild-type ERβ (ERβ1) may not be adequate in explaining the still controversial role of ERβ(s) in prostate carcinogenesis. In this study, using newly generated isoform-specific antibodies, immunohistochemistry (IHC) was performed on a tumor microarray comprised of 144 specimens. IHC results were correlated with pathological and clinical follow-up data to delineate the distinct roles of ERβ1, ERβ2, and ERβ5 in PCa. ERβ2 was commonly found in the cytoplasm and was the most abundant isoform followed by ERβ1 localized predominantly in the nucleus, and ERβ5 was primarily located in the cytoplasm. Logistic regression analyses demonstrated that nuclear ERβ2 (nERβ2) is an independent prognostic marker for prostate specific antigen (PSA) failure and postoperative metastasis (POM). In a Kaplan–Meier analysis, the combined expression of both nERβ2 and cytoplasmic ERβ5 identified a group of patients with the shortest POM-free survival. Cox proportional hazard models revealed that nERβ2 predicted shorter time to POM. In concordance with IHC data, stable, ectopic expression of ERβ2 or ERβ5 enhanced PCa cell invasiveness but only PCa cells expressing ERβ5 exhibited augmented cell migration. This is the first study to uncover a metastasis-promoting role of ERβ2 and ERβ5 in PCa, and show that the two isoforms, singularly and conjointly, have prognostic values for PCa progression. These findings may aid future clinical management of PCa

Environmental factors, epigenetics, and developmental origin of reproductive disorders

Sex-specific differentiation, development, and function of the reproductive system are largely dependent on steroid hormones. For this reason, developmental exposure to estrogenic and anti-androgenic endocrine disrupting chemicals (EDCs) is associated with reproductive dysfunction in adulthood. Human data in support of “Developmental Origins of Health and Disease” (DOHaD) comes from multigenerational studies on offspring of diethylstilbestrol-exposed mothers/grandmothers. Animal data indicate that ovarian reserve, female cycling, adult uterine abnormalities, sperm quality, prostate disease, and mating behavior are susceptible to DOHaD effects induced by EDCs such as bisphenol A, genistein, diethylstilbestrol, p,p′-dichlorodiphenyl-dichloroethylene, phthalates, and polyaromatic hydrocarbons. Mechanisms underlying these EDC effects include direct mimicry of sex steroids or morphogens and interference with epigenomic sculpting during cell and tissue differentiation. Exposure to EDCs is associated with abnormal DNA methylation and other epigenetic modifications, as well as altered expression of genes important for development and function of reproductive tissues. Here we review the literature exploring the connections between developmental exposure to EDCs and adult reproductive dysfunction, and the mechanisms underlying these effects