Oral exposure to methylmercury modifies the prostatic microenvironment in adult rats

Methylmercury (MeHg) is an environmental pollutant that is highly toxic to the central nervous system. As its effects on male reproductive system are poorly understood, this study was carried out to analyse the effects of MeHg on the rat prostate. To evaluate the MeHg toxicity on ventral prostate, three groups of adult male Wistar rats received oral doses of 0.5, 1.0 and 3.0mg/kg MeHg, respectively, on a daily basis for 14days. A fourth group was used as a control. The prostate weight was decreased in rats treated orally with 0.5mg/kg MeHg compared to controls. Also, Hg concentration increased significantly in the prostate after treatments. There were reductions in serum testosterone levels and androgen receptor immunoreactivity in animals receiving 3.0mgMeHg/kg. The stereological data showed changes in the prostatic epithelial, stromal and luminal compartments which varied according to the different doses. Histopathological alterations, such as chronic inflammation, stratified epithelial hyperplasia and epithelial inflammatory reactive atypia, were observed in the 0.5mg/kg MeHg-treated group. Epithelial atrophy was observed in the 3.0mg/kg MeHg-treated group. In conclusion, the MeHg affects prostatic homoeostasis resulting in histopathological changes that may be relevant in the pathogenesis of prostatic disease.

Impairment on sperm quality and fertility of adult rats after antiandrogen exposure during prepuberty

This study evaluated the effects of antiandrogen exposure during the prepubertal period on reproductive development and reproductive competence in adults. Male rats were divided into two groups: flutamide, receiving 25 mg/kg/day of flutamide by oral gavage and control, receiving vehicle daily. Dosing continued from PND 21 to 44, and animals were killed on PND 50 or PND 75-80. The epididymis, prostate, vas deferens and seminal vesicle weights were lower in Flutamide group on PND 50, while on PND 80 only seminal vesicle weight was reduced. Fertility assessed by IUI revealed a decrease in the fertility potential in the flutamide-treated adults. Flutamide accelerated sperm transit time through the epididymis, impairing sperm motility and storage. A quantitative analysis of the cauda sperm membrane proteome revealed a few significant changes in protein expression. Thus, exposure to flutamide during the prepubertal period compromises the function of the epididymis along with epididymal sperm quality at adulthood. (C) 2011 Elsevier Inc. All rights reserved.

Cyclosporin A causes impairment of the ventral prostate tissue structure of Wistar rats

Cyclosporin A (CsA) is an immunosuppressive drug widely used in medicine to reduce the immune system activity and, therefore, the risk of organ rejection after transplantation. However, many side effects can be related to its use, such as, reduction in serum testosterone levels due to damage of the testis structure and, consequently, male infertility. The present study aims to evaluate the effects of chronic CsA administration on the ventral prostate tissue ( 1 5 mg/kg per d, for 56 days). Stereological, morphometrical, morphological and ultrastructural observations were employed. The plasmatic testosterone and glucose levels were measured. An androgen receptor (AR) immunohistochemical method was applied on ventral prostate sections. Apoptosis was detected with the terminal deoxynucleotidyl transferase dUTP nick end labeling technique. CsA treatment caused reduction in plasmatic testosterone levels and an increase in glycemia. The volume of all ventral prostate tissue components (lumen, epithelium and muscular and nonmuscular stroma) and ventral prostate weight were reduced in the CsA-treated group. Light and transmission electron microscopy confirmed epithelium atrophy of treated animals. There was no alteration of AR expression or apoptotic index. CsA chronic treatment in the therapeutic doses caused damage to prostate tissue of adult Wistar rats, probably due to increase in the glucose levels and reduction in the plasmatic testosterone levels.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Abstract Background Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods Twenty-four adult Wistar rats, 60 days old (+/- 250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [100 μg/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a.m. Results Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

MicroRNAs y receptores hormonales: identificación y cuantificación en tumores de mama

[ES] En este trabajo, hemos tratado de identificar, y poner a punto las técnicas necesarias para la cuantificación de los miRNAs asociados con el control post-transcripcional del receptor de andrógenos (AR) y el receptor de estrógenos (ER) en tumores de mama. Para ello, hemos usado las bases de datos publicadas en Internet. Además, comparamos la cantidad y la calidad del RNA total aislado (miRNA y mRNA) en 11 tumores de mama incluidos en parafina mediante el uso de dos kits comerciales.

Genomic and non genomic effects of elevated concentration of anabolic steroids in human neuronal cells

Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter the expression and function of neurotransmitter systems and contribute to neuronal cell death. This effect can explain the behavioural changes, drug dependence and neuro degeneration observed in steroid abuser. Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through post-transcriptional mechanisms requiring the AR. Cito-toxicity assays demonstrated a time- and concentration dependent decrease of cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was observed in cells treated with Nandrolone 10-6M for 48h. Collectively, these data support the existence of two cellular mechanisms that might explain the neurological syndromes observed in steroids abuser.

Sintesi di nuovi derivati tri-componente per target photodynamic therapy della neoplasia prostatica

Therapies for the treatment of prostate cancer show several limitations, especially when the cancer metastasizes or acquires resistance to treatment. In addition, most of the therapies currently used entails the occurrence of serious side effects. A different therapeutic approach, more selective and less invasive with respect either to radio or to chemotherapy, is represented by the photodynamic therapy (PDT). The PDT is a treatment that makes use of photosensitive drugs: these agents are pharmacologically inactive until they are irradiated with light at an appropriate wavelength and in the presence of oxygen. The drug, activated by light, forms singlet oxygen, a highly reactive chemical species directly responsible for DNA damage, thus of cell death. In this thesis we present two synthetic strategies for the preparation of two new tri-component derivatives for photodynamic therapy of advanced prostate cancer, namely DRPDT1 and DRPDT2. Both derivatives are formed by three basic elements covalently bounded to each other: a specific ligand with high affinity for the androgen receptor, a suitably chosen spacer molecule and a photoactivated molecule. In particular, DRPDT2 differs from DRPDT1 from the nature of the AR ligand. In fact, in the case of DRPDT2 we used a synthetically engineered androgen receptor ligand able to photo-react even in the absence of oxygen, by delivering NO radical. The presence of this additional pharmacophore, together with the porphyrin, may ensure an additive/synergistic effect to the photo-stimulated therapy, which than may act both in the presence of oxygen and in hypoxic conditions. This approach represents the first example of multimodal photodynamic therapy for prostate cancer.

Effetti della somministrazione di Testosterone in donne

Come noto, il testosterone (T) gioca un ruolo importante in differenti funzioni fisiologiche. Il ruolo del T nelle donne è tuttavia largamente sconosciuto. Recenti studi riportano un ruolo del T nella modulazione della funzionalità sessuale femminile. SCOPO: Indagare gli effetti del T nelle donne, su parametri metabolici, ossei e composizione corporea e studiare gli effetti del T sulla proliferazione e innervazione della vagina. METODI: 16 soggetti FtM ovariectomizzati sono stati sottoposti a terapia con TU 1000 mg im + placebo o dutasteride. Alla settimana 0 e 54 sono stati valutati: parametri metabolici e composizione corporea. 16 campioni di tessuto vaginale ottenuti da soggetti FtM trattati con T, 16 donne PrM e 16 donne M sono stati analizzati. Sono stati valutati: morfologia, contenuto di glicogeno, espressione del Ki-67, recettori per estrogeni e androgeni ed innervazione. RISULTATI: La somministrazione di T in soggetti FtM determina aumento del colesterolo LDL e riduzione delle HDL. L’HOMA si riduce significativamente nel gruppo TU e tende ad aumentare nel gruppo TU+D. L’ematocrito aumenta. BMI, WHR e grasso tendono a ridursi, la massa magra ad aumentare. Non riportiamo cambiamenti del metabolismo osseo. Nel tessuto vaginale di FtM osserviamo perdita della normale architettura dell’epitelio. La somministrazione di T determina riduzione della proliferazione cellulare. I recettori per E e il PGP 9.5 sono significativamente ridotti nei FtM. La presenza di recettori per A è dimostrata nello stroma e nell’epitelio. L’espressione di AR si riduce con l’età e non cambia con la terapia con T nella mucosa, mentre aumenta nello stroma dopo somministrazione di T. CONCLUSIONI: Non riportiamo effetti avversi maggiori dopo somministrazione di T. La terapia con T determina ridotta proliferazione dell’epitelio vaginale. I recettori per AR sono presenti sia nello stroma che nell’epitelio. T aumenta l’espressione di AR nello stroma.

Small Molecules as Modulators of Different Targets Involved in Tumor Progression

Tumor is a lesion that may be formed by an abnormal growth of neoplastic cells. Many factors increase the risk of cancer and different targets are involved in tumor progression. Within this thesis, we have addressed two different biological targets, independently connected with tumor formation, e.g. Hsp90 and androgen receptor. The ATP-dependent chaperone Hsp90 is responsible for the conformational maturation and the renaturation of proteins. “Client” proteins are associated with the cancer hallmarks, as cell proliferation and tumor progression. Consequently, Hsp90 has evolved into promising anticancer target. Over the past decade, radicicol has been identified as potential anticancer agent targeting Hsp90, but it is not active in vivo. With that aim of obtaining radicicol-related derivatives, we developed the design and synthesis of new chalcones analogs. Chalcones, which are abundant in edible plants, own a diverse array of pharmacological activities and are considered a versatile scaffold for drug design. Antiproliferative assays and western blot analysis on the new compounds showed that some of those display an interesting cytotoxic effect and the ability to modulate Hsp90 client proteins expression. Androgen Receptor (AR) hypersensitivity plays crucial role in prostate cancer, which progression is stimulated by androgens. The therapy consists in a combination of surgical or chemical castration, along with antiandrogens treatment. Casodex® (bicalutamide), is the most widespread antiandrogen used in clinic. However, hormonal therapy is time-limited since many patients develop resistance. Commercially available antiandrogens show a common scaffold, e.g. two substituted aromatic rings linked by a linear or a cyclic spacer. With the aim of obtaining novel pure AR antagonists, we developed a new synthetic methodology, which allowed us to introduce, as linker between two suitably chosen aromatic rings, a triazole moiety. Preliminary data suggest that the herein reported new molecules generally decrease PSA expression, thus confirming their potential AR antagonistic activity.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice

Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice.

Yolk Androstenedione in Domestic Chicks (Gallus Gallus Domesticus): Uptake and Sex-Dependent Alteration of Growth and Behavior

In birds, causes and consequences of variation in maternally-derived steroids in egg yolk have been the subject of intense experimentation. Many studies have quantified or manipulated testosterone ("T") and one of its immediate precursors, androstenedione ("A4") - often lumping the two steroids as "androgens" and treating them as functionally equivalent. However, yolk A4 is deposited in substantially higher concentrations than T, binds only weakly to the androgen receptor, and is readily converted into either T or estrone by steroidogenic enzymes present during embryonic development. Thus it may not be appropriate to assume that A4 has the same effect as T. In addition, A4's metabolic fate is likely to differ between females and males. The goals of this study were to examine the sex-specific uptake and metabolism of yolk A4 and consequences of elevated levels of yolk A4 on development and behavior of domestic chicks. Eggs were injected with 2mu Ci of tritiated androstenedione; radioactivity was detected in all tissues of day 7 and day 16 embryos and found in both aqueous and organics phases of day 7 yolk, with no difference between sexes. A second set of eggs was injected with 125ng of A4. A4 increased growth of morphological traits (tarsus, beak) in females, but not males. A4 males had smaller combs than controls; there was no treatment effect in females. A4 reduced tonic immobility behavior in both sexes. The results of this study illustrate the importance of distinguishing both between androgens and between sexes when investigating avian endocrine maternal effects. Copyright 2013 Elsevier Inc. All rights reserved.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing, CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, pre-existing SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SC-like BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (AR(low)) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The AR(low) seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

[How should hormone therapy for castration-resistant prostate cancer be continued?]

After an average of 18-36 months under androgen suppression therapy by surgical castration, LHRH, and steroidal or non-steroidal antiandrogens, almost all patients with metastatic prostate cancer show PSA progression as a sign of androgen-independent but still androgen-sensitive tumor growth. Our understanding and the treatment of such castration-resistant prostate cancer has changed markedly. The introduction of new drugs like abiraterone and MDV3100 has shown that prostate cancer progression even in the"hormone-refractory" stage is driven by androgen receptor signaling. Based on this information the question of whether androgen deprivation therapy in castration-resistant prostate cancer should be continued or not is still of relevance. This review gives a critical overview of the literature and current guideline recommendations.

Significance of Increased Tissue Transglutaminase in Hormone Refractory Prostate Cancer

The progression of hormone responsive to hormone refractory prostate cancer poses a major clinical challenge in the successful treatment of prostate cancer. The hormone refractory prostate cancer cells exhibit resistance not only to castrate levels of testosterone, but also to other therapeutic modalities and hence become lethal. Currently, there is no effective treatment available for managing this cancer. These observations underscore the urgency to investigate mechanism(s) that contribute to the progression of hormone-responsive to hormone-refractory prostate cancer and to target them for improved clinical outcomes. Tissue transglutaminase (TG2) is a multifunctional pro-inflammatory protein involved in diverse physiological processes such as inflammation, tissue repair, and wound healing. Its expression is also implicated in pathological conditions such as cancer and fibrosis. Interestingly, we found that the androgen-independent prostate cancer cell lines, which lacked androgen receptor (AR) expression, contained high basal levels of tissue transglutaminase. Inversely, the cell lines that expressed androgen receptor lacked transglutaminase expression. This attracted our attention to investigate the possible role this protein may play in the progression of prostate cancer, especially in view of recent observations that its expression is linked with increased invasion, metastasis, and drug resistance in multiple cancer cell types. The results we obtained were rather surprising and revealed that stable expression of tissue transglutaminase in androgen-sensitive LNCaP prostate cancer cells rendered these cells independent of androgen for growth and survival by silencing the AR expression. The AR silencing in TG2 expressing cells (TG2-infected LNCaP and PC-3 cells) was due to TG2-induced activation of the inflammatory nuclear transcription factor-kB (NF-kB). Thus, TG2 induced NF-kB was found to directly bind to the AR promoter. Importantly, TG2 protein was specifically recruited to the AR promoter in complex with the p65 subunit of NF-kB. Moreover, TG2 expressing LNCaP and PC-3 cells exhibited epithelial-to-mesenchymal transition, as evidenced by gain of mesenchymal (such as fibronectin, vimentin, etc.) and loss of epithelial markers (such as E-cadherin, b-catenin). Taken together, these results suggested a new function for TG2 and revealed a novel mechanism that is responsible for the progression of prostate cancer to the aggressive hormone-refractory phenotype.

Genetic predisposition to prostate cancer

Prostate cancer (PC) is a significant economic and health burden in the U.S. and Europe but its causes are largely unknown. The most significant risk factors (after gender) are age and family history of the disease. A gene with high penetrance but low frequency on chromosome 1q, HPC 1, has been suggested to cause a proportion of the familial aggregation of PC but other more common genes, conferring less risk, are also thought to contribute to disease predisposition. We have pursued a strategy to study both types of genetic risk in PC. To identify high penetrance genes, affected men from thirteen families have been genotyped for genetic linkage analysis at six microsatellite markers spanning 45 cM of 1q24-25. Both LOD score and non-parametric statistics provide no significant support for HPC1 in this genomic region, although 3 of the families did combine to produce a LOD score of 0.9. These families will be included in a genome wide search for other PC predisposition genes as part of a multinational collaboration.^ For study of common genetic factors in PC development, leukocyte DNA samples from an unselected series of 55 patients and 67 controls have been examined for genetic differences in two other candidate genes, the androgen receptor gene, hAR, at Xq11-12, and the vitamin D receptor gene, hVDR, at 12q12-14. hAR was typed for two trinucleotide repeat length polymorphisms, (CAG)$\rm\sb{n}$ and (GGC)$\rm\sb{n},$ encoding polyglutamine and polyglycine tracts, respectively, which have been implicated in PC susceptibility. These data, combined with similarly processed patients and controls from the U.K. show no consistent association of allele length with PC risk. A novel finding, however, has been a significant association between the number of GGC repeats and the length of time between diagnosis and relapse in stage T1-T4 Caucasian patients irrespective of therapy and age of the patient. Of 49 patients who relapsed out of 108 entering the study, those with 16 or fewer GGC repeats had an average relapse-free-period of 101 (+/$-$7.7) months while for those with more than 16 repeats the period averaged 48 (+/$-$2.9) months, a difference of 2.1 fold or 4.4 years.^ The second gene, hVDR, was genotyped at two polymorphisms, a synonymous C/T substitution in exon 9 identified by differential TaqI enzymatic digestion and a variable length polyA tract in the 3$\sp\prime$ UTR. Although these polymorphisms are in strong linkage disequilibrium only the polyA region showed a possible association with PC risk. Men homozygous for alleles with fewer than 18 A's had an increased risk (OR = 3.0, p = 0.0578) compared to controls. This result is opposite to the findings of others and may either indicate off-setting random errors which together balance out to no significant overall effect or reflect more complex genetic and/or environmental associations.^ Overall, this research suggests that single gene familial predisposition may be less prominent in PC than in other cancers and that the characteristics of PC pathology may be useful in identifying the effects of common genetic factors. ^