Parallel targeted and non-targeted quantitative analysis of steroids in human serum and peritoneal fluid by liquid chromatography high-resolution mass spectrometry.

We developed and validated a liquid chromatography high-resolution mass spectrometry method for the absolute quantification of 51 steroids for clinical analysis of human serum and, for the first time, peritoneal fluid. Data acquisition was performed in both targeted and untargeted mode simultaneously, thus allowing the accurate and precise quantification of the main components of the classical steroid pathways (17 steroids) as well as the analysis of 34 additional non-classical steroids. For targeted analysis, validation was performed according to FDA guidelines, resulting, among other parameters, in accuracy < 13% RSD and precision < 10% relative error, for both inter- and intra-day validation runs. By establishing steroid-specific response factors, the calibration curves of the targeted analytes can be extended to untargeted analytes. This approach opens novel possibilities for the post hoc analysis of clinical samples as the data can be examined for virtually any steroid even after data acquisition, enabling facile absolute quantification once a standard becomes available. We demonstrate the applicability of the approach to evaluate the differences in steroid content between peripheral serum and peritoneal fluid across the menstrual cycle phases, as well as the effect of the synthetic gestagen dienogest on the steroid metabolome.

18F-RB390: innovative ligand for imaging the T877A androgen receptor mutant in prostate cancer via positron emission tomography (PET).

BACKGROUND: Detecting prostate cancer before spreading or predicting a favorable therapy are challenging issues for impacting patient's survival. Presently, 2-[(18) F]-fluoro-2-deoxy-D-glucose ((18) F-FDG) and/or (18) F-fluorocholine ((18) F-FCH) are the generally used PET-tracers in oncology yet do not emphasize the T877A androgen receptor (AR) mutation being exclusively present in cancerous tissue and escaping androgen deprivation treatment. METHODS: We designed and synthesized fluorinated 5α-dihydrotestosterone (DHT) derivatives to target T877A-AR. We performed binding assays to select suitable candidates using COS-7 cells transfected with wild-type or T877A AR (WT-AR, T877A-AR) expressing plasmids and investigated cellular uptake of candidate (18) F-RB390. Stability, biodistribution analyses and PET-Imaging were assessed by injecting (18) F-RB390 (10MBq), with and without co-injection of an excess of unlabeled DHT in C4-2 and PC-3 tumor bearing male SCID mice (n = 12). RESULTS: RB390 presented a higher relative binding affinity (RBA) (28.1%, IC50 = 32 nM) for T877A-AR than for WT-AR (1.7%, IC50 = 357 nM) related to DHT (RBA = 100%). A small fraction of (18) F-RB390 was metabolized when incubated with murine liver homogenate or human blood for 3 hr. The metabolite of RB390, 3-hydroxysteroid RB448, presented similar binding characteristics as RB390. (18) F-RB390 but not (18) F-FDG or (18) F-FCH accumulated 2.5× more in COS-7 cells transfected with pSG5AR-T877A than with control plasmid. Accumulation was reduced with an excess of DHT. PET/CT imaging and biodistribution studies revealed a significantly higher uptake of (18) F-RB390 in T877A mutation positive xenografts compared to PC-3 control tumors. This effect was blunted with DHT. CONCLUSION: Given the differential binding capacity and the favorable radioactivity pattern, (18) F-RB390 represents the portrayal of the first imaging ligand with predictive potential for mutant T877A-AR in prostate cancer for guiding therapy. Prostate 75:348-359, 2015. © 2014 Wiley Periodicals, Inc.

Association of endocannabinoids with pain in endometriosis.

ABSTRACT: Endocannabinoid (eCB) levels fluctuate in inflammatory conditions and as such may take part in endometriosis-associated pain or even in endometriosis pathogenesis. In this case-control (23 cases and 19 controls) study, targeted lipids were measured in the serum and peritoneal fluid collected during laparoscopy. Endometriosis was confirmed histologically. Dysmenorrhea, abdominal pain, and dyspareunia were assessed using the Numeric Rating Scale for pain. Steroids, eCBs, and related lipids were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tumor necrosis factor alpha, IL-8, PAPP-A, PP14, RANTES, OPG, MIDKINE, MCP-1, VEGF, leptin, and defensins were quantified by ELISA. We found that eCB levels were significantly influenced by both noncyclic and cyclic abdominal pain. Specifically, women suffering from noncyclic abdominal pain were characterized by a higher 2-AG level in the peritoneal fluid throughout the menstrual cycle, whereas women suffering from dysmenorrhea had higher 2-AG levels and lower AEA levels during the proliferative phase alone. In addition, 2-AG positively correlated with prostaglandin E2 (PGE2), and the ratio AEA/2-AG positively correlated with defensins, suggesting a possible link between endocannabinoids system and inflammatory pain. The results of the current study indicate that the eCB system may play a role in endometriosis-associated pain, but additional studies are needed to investigate the causal relationship.

Epithelial-to-mesenchymal transition contributes to the downregulation of progesterone receptor expression in endometriosis lesions.

Endometriosis is a common, estrogen-dependent disease, in which endometrial tissue grows in the peritoneal cavity. These lesions often express low levels of progesterone receptors (PR), which potentially play an important role in the insufficient response to progestin treatment. Here, we uncover an interconnection between the downregulated PR expression and the epithelial-to-mesenchymal transition (EMT) in endometriotic lesions. The majority of ectopic epithelial glands (93.1 %, n = 67/72) display heterogeneous states of EMT by immunohistochemistry staining. Interestingly, low PR expression associated with high N-cadherin expression, a hallmark of EMT. In order to gain mechanistic insights, we performed in vitro functional assays with the endometriotic epithelial cell lines EM'osis and 12Z. TGF-ß-induced EMT, marked by elevations of CDH2 and SNAI1/2, led to a significant downregulation of PR gene expression in both cell lines. In contrast, silencing of SNAI1 in EM'osis and of SNAI1 plus SNAI2 in 12Z elevated PR gene expression significantly. We found that not only in vitro, but also in the epithelial component of endometriotic lesions strong expression of SNAI1/2 concurred with weak expression of PR. In summary, these results suggested the negative correlation association of the heterogeneous states of EMT and suppressed PR expression in endometriotic lesions. Our functional assays indicate that EMT contributes to the downregulation of PR expression via the upregulation of EMT-TFs, like SNAI1 and SNAI2, which may ultimately lead to progesterone resistance.

The absence of a functional nuclear receptor element A (NREA) in the promoter II of the aromatase P450 gene in rabbit granulosa cells.

Aromatase protein is synthesized in response to gonadotropins that activate expression of their target genes via the cAMP second messenger system. The -882/+103 bp region of the rabbit ovarian promoter (PII) was ligated to a luciferase vector and transfected into granulosa cells to elucidated the mechanism by which cAMP stimulates transcription. Deletions and mutational experiments indicate that (i) a cAMP-response element-like sequence (CLS) present at -208 to -200 bp is the main element required for the activation of the rabbit PII by cAMP and that (ii) both nuclear receptor element sites; NREA (-133/-126 bp) and NREB (-188/-181 bp) do not participate to the cAMP-dependent activity of the PII. The replacement of the specific rabbit NREA site by the human NREA site increases two-fold the cAMP response and indicates that trans-activating factors are present in rabbit granulosa cells. This study shows for the first time an efficient aromatase transcription occurs in granulosa cells in absence of a consensus NREA site. In addition a comparative study has been performed on the sheep aromatase promoter where sites deviate from rabbit. Mutagenesis experiments suggest that some of them are involved in the cAMP-induced response of the rabbit PII.

Detection and functional portrayal of a novel class of dihydrotestosterone derived selective progesterone receptor modulators (SPRM).

In early pregnancy, abortion can be induced by blocking the actions of progesterone receptors (PR). However, the PR antagonist, mifepristone (RU38486), is rather unselective in clinical use because it also cross-reacts with other nuclear receptors. Since the ligand-binding domain of human progesterone receptor (hPR) and androgen receptor (hAR) share 54% identity, we hypothesized that derivatives of dihydrotestosterone (DHT), the cognate ligand for hAR, might also regulate the hPR. Compounds designed and synthesized in our laboratory were investigated for their affinities for hPRB, hAR, glucocorticoid receptor (hGRα) and mineralocorticoid receptor (hMR), using whole cell receptor competitive binding assays. Agonistic and antagonistic activities were characterized by reporter assays. Nuclear translocation was monitored using cherry-hPRB and GFP-hAR chimeric receptors. Cytostatic properties and apoptosis were tested on breast cancer cells (MCF7, T-47D). One compound presented a favorable profile with an apparent neutral hPRB antagonistic function, a selective cherry-hPRB nuclear translocation and a cytostatic effect. 3D models of human PR and AR with this ligand were constructed to investigate the molecular basis of selectivity. Our data suggest that these novel DHT-derivatives provide suitable templates for the development of new selective steroidal hPR antagonists.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11ß-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11ß-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.

Regulation of 11ß-hydroxysteroid dehydrogenase type 2 by microRNA.

The enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is selectively expressed in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor. A diminished activity causes salt-sensitive hypertension. The mechanism of the variable and distinct 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) expression in the cortical collecting duct is poorly understood. Here, we analyzed for the first time whether the 11ß-HSD2 expression is modulated by microRNAs (miRNAs). In silico analysis revealed 53 and 27 miRNAs with potential binding sites on human or rat HSD11B2 3'-untranslated region. A reporter assay demonstrated 3'-untranslated region-dependent regulation of human and rodent HSD11B2. miRNAs were profiled from cortical collecting ducts and proximal convoluted tubules. Bioinformatic analyses showed a distinct clustering for cortical collecting ducts and proximal convoluted tubules with 53 of 375 miRNAs, where 13 were predicted to bind to the rat HSD11B2 3'-untranslated region. To gain insight into potentially relevant miRNAs in vivo, we investigated 2 models with differential 11ß-HSD2 activity linked with salt-sensitive hypertension. (1) Comparing Sprague-Dawley with low and Wistar rats with high 11ß-HSD2 activity revealed rno-miR-20a-5p, rno-miR-19b-3p, and rno-miR-190a-5p to be differentially expressed. (2) Uninephrectomy lowered 11ß-HSD2 activity in the residual kidney with differentially expressed rno-miR-19b-3p, rno-miR-29b-3p, and rno-miR-26-5p. In conclusion, miRNA-dependent mechanisms seem to modulate 11ß-HSD2 dosage in health and disease states.

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation.

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

A novel steroidal antiandrogen targeting wild type and mutant androgen receptors.

Prostate cancer (PCa) progression is enhanced by androgen and treatment with antiandrogens represents an alternative to castration. While patients initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years by expressing androgen receptor (AR) mutants. Such mutations, indeed, promote unfavorable agonistic behavior from classical antagonists. Here, we have synthesized and screened 37 novel compounds derived from dihydrotestosterone (DHT), cyanolutamide and hydroxyflutamide. These derivatives were tested for their potential antagonistic activity using a luciferase reporter gene assay and binding properties were determined for wild type (WT) and mutant ARs (T877A, W741C, W741L, H874Y). In the absence and presence of antiandrogens, androgen dependent cellular proliferation and prostate specific antigen (PSA) expression were assayed in the prostate cancer cell line LNCaP by crystal violet, real time PCR and by Western blots. Also, cellular proliferation and PSA expression were assayed in 22Rv1. A novel compound RB346, derived from DHT, was found to be an antagonist for all tested AR forms, preventing DHT induced proliferation and PSA expression in LNCaP and 22Rv1 cells. RB346 displayed no agonistic activity, in contrast to the non-steroidal antiandrogen bicalutamide (Casodex) with unfavorable agonistic activity for W741L-AR. Additionally, RB346 has a slightly higher binding affinity for WT-AR, T877A-AR and H874Y-AR than bicalutamide. Thus, RB346 is the first potent steroidal antiandrogen with efficacy for WT and various AR mutants.

cAMP-dependent regulation of CYP19 gene in rabbit preovulatory granulosa cells and corpus luteum.

Transcription of the CYP19 gene encoding the aromatase P450 enzyme in ovarian cells is under the control of the two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), via modulation of intracellular cyclic 3',5'-adenosine monophosphate (cAMP) levels. Primary cultures of rabbit ovarian cells were used to identify the functional regions of the ovarian promoter (PII) that are responsive to the gonadotropic secondary messenger and to estradiol. Transfection experiments in granulosa and luteal cells with deleted constructs of the PII promoter show that the region between -274 and -193bp is critical for cAMP-dependent transcriptional activity. A comparison of PII activities in granulosa and small luteal cells highlights a 50% decrease consecutive to the LH surge. Sequence analysis of the above mentioned region revealed the presence of a cAMP responsive element like sequence (CLS) and of a nuclear receptor element A (NREA). Binding of CREB to CLS has been shown using granulosa and luteal cells nuclear extracts. In addition, we identified the expression of NR5A1 (Steroidogenic Factor 1) and NR5A2 (Liver Receptor Homologue 1) in granulosa and luteal cells. However, the binding to NREA is observed only with granulosa cells nuclear extracts. Data suggest that the NR5A factors are not the main regulators of CYP19 gene, in contrast with the others genes of streroidogenesis enzymes, and additional sites may play an important role during the post-LH surge down-regulation of CYP19 transcription.