A selective alpha1D-adrenoreceptor antagonist inhibits human prostate cancer cell proliferation and motility "in vitro".

The progression of prostate cancer (PC) to a metastatic hormone refractory disease is the major contributor to the overall cancer mortality in men, mainly because the conventional therapies are generally ineffective at this stage. Thus, other therapeutic options are needed as alternatives or in addition to the classic approaches to prevent or delay tumor progression. Catecholamines participate to the control of prostate cell functions by the activation of alpha1-adrenoreceptors (alpha1-AR) and increased sympathetic activity has been linked to PC development and evolution. Molecular and pharmacological studies identified three alpha1-AR subtypes (A, B and D), which differ in tissue distribution, cell signaling, pharmacology and physiological role. Within the prostate, alpha1A-ARs mainly control stromal cell functions, while alpha1B- and alpha1D- subtypes seem to modulate glandular epithelial cell growth. The possible direct contribution of alpha1D-ARs in tumor biology is supported by their overexpression in PC. The studies here presented investigate the "in vitro" antitumor action of A175, a selective alpha1D-AR antagonist we have recently obtained by modifying the potent, but not subtype-selective alpha1-AR antagonist (S)-WB4101, in the hormone-refractory PC3 and DU145 PC cell lines. The results indicate that A175 has an alpha1D-AR-mediated significant and dose-dependent antiproliferative action that possibly involves the induction of G0/G1 cell cycle arrest, but not apoptosis. In addition, A175 reduces cell migration and adhesiveness to culture plates. In conclusion, our work clarified some cellular aspects promoted by alpha1D-AR activity modulation and supports a further pharmacological approach in the cure of hormone-refractory PC, by targeting specifically this AR subtype.

Learning and memory: Steroids and epigenetics.

Memory formation and utilization is a complex process involving several brain structures in conjunction as the hippocampus, the amygdala and the adjacent cortical areas, usually defined as medial temporal lobe structures (MTL). The memory processes depend on the formation and modulation of synaptic connectivity affecting synaptic strength, synaptic plasticity and synaptic consolidation. The basic neurocognitive mechanisms of learning and memory are shortly recalled in the initial section of this paper. The effect of sex hormones (estrogens, androgens and progesterone) and of adrenocortical steroids on several aspects of memory processes are then analyzed on the basis of animal and human studies. A specific attention has been devoted to the different types of steroid receptors (membrane or nuclear) involved and on local metabolic transformations when required. The review is concluded by a short excursus on the steroid activated epigenetic mechanisms involved in memory formation.

Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton.

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30-150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.

In vitro chronic administration of ERbeta selective ligands and prostate cancer cell growth: hypotheses on the selective role of 3beta-adiol in AR-positive RV1 cells.

Prostate cancer (PC) progression from androgen-dependent (AD) to castration-resistant (CR) disease is a process caused by modifications of different signal transduction pathways within tumor microenvironment. Reducing cell proliferation, estrogen receptor beta (ERbeta) is emerging as a potential target in PC chemoprevention. Among the known selective ERbeta ligands, 3beta-Adiol, the endogenous ligand in the prostate, has been proved to counteract PC progression. This study compares the effects of chronic exposure (1-12 weeks) to different ERbeta selective ligands (DPN, 8beta-VE2, 3beta-Adiol) on proliferation of human androgen-responsive CWR22Rv1 cells, representing an intermediate phenotype between the AD- and CR-PC. 3beta-Adiol (10 nM) is the sole ligand decreasing cell proliferation and increasing p21 levels. In vitro transcriptional activity assays were performed to elucidate different behavior between 3beta-Adiol and the other ligands; in these experiments the endogenous and the main ERbeta subtype activation were considered. It is concluded that ERbeta activation has positive effects also in androgen-responsive PC. The underlying mechanisms are still to be clarified and may include the interplay among different ERbeta subtypes and the specific PC microenvironment. ERbeta agonists might be useful in counteracting PC progression, although the final outcome may depend upon the molecular pattern specific to each PC lesion.

Androgen receptor activation by polychlorinated biphenyls: epigenetic effects mediated by the histone demethylase Jarid1b.

The exposure to environmental endocrine disrupting compounds (EDC), as polychlorinated biphenyls (PCBs), widely diffused in the environment may produce epigenetic changes that affect the endocrine system. We found that PCBs activate AR transcriptional activity and that this effect is potentiated by the demethylase Jarid1b, a histone demethylase that catalyzes the removal of trimethylation of lysine 4 on histone H3 (H3K4me3), induced by PCB. The aim of the present study was to investigate the effect of the treatment of cultured cells (HEK293) with a mixture of the most diffused environmental PCBs and, also with dihydrotestosterone (DHT), on the functional interaction between AR and Jarid1b. Although the effect induced by DHT on the AR transactivation was considerably higher, the PCB mixture produced an AR-mediated transactivation in a dose-dependent manner. Cotransfection with plasmids expressing Jarid1b and various AR isoforms containing polyglutamine tracts (polyQ tracts) of different lengths showed that Jarid1b potentiates the AR transcriptional activity induced by PCBs but only with the shortest AR isoform. The potentiating effect of Jarid1b on the AR is mediated by a direct interaction of the enzyme with the AR promoter. In fact, utilizing constructs containing AR promoters with a different length and a luciferase reporter gene, we showed that the effect of PCBs, but not of DHT, needs the presence of Jarid1b and of at least two DNA binding sites for Jarid1b.

PXD101 potentiates hormonal therapy and prevents the onset of castration-resistant phenotype modulating androgen receptor, HSP90, and CRM1 in preclinical models of prostate cancer.

Aberrant activation or 'reactivation' of androgen receptor (AR) during androgen ablation therapy shows a potential cause for the development of castration-resistant prostate cancer. This study tested the hypothesis that PXD101, a potent pan histone deacetylase (HDAC) inhibitor, may prevent onset of castration-resistant phenotype and potentiate hormonal therapy. A panel of human prostate cancer cells with graded castration-resistant phenotype and in vivo models were used to verify this hypothesis. In this report, we demonstrated that hormonal manipulation favors the onset of castration-resistant phenotype increasing HDAC expression and activity as well as modulating expression and activity of AR, EGFR, HER2, and Akt. Consistent with these observations, the functional knockdown of HDACs by PXD101 prevented the onset of castration-resistant phenotype with a significant downregulation of AR, EGFR, HER2, and Akt expression/activity. The dysregulation of functional cooperation between HDAC6 with hsp90, on the one hand, and between GSK-3ß with CRM1, on the other hand, may explain the biological effects of PXD101. In this regard, the HDAC6 silencing or the functional knockdown of hsp90 by 17AAG resulted in the selective downregulation of AR, EGFR, HER2, and Akt expression/activity, while the decreased phosphorylation of GSK-3ß mediated by PXD101 increased the nuclear expression of CRM1, which in turn modified the AR and survivin recycling with increased caspase 3 activity. HDAC inhibitors retain the ability to prevent the onset of castration-resistant phenotype and, therefore, merit clinical investigation in this setting. However, additional data are needed to develop clinical treatment strategies for this disease stage.

Polychlorinated biphenyls affect histone modification pattern in early development of rats: a role for androgen receptor-dependent modulation?

BACKGROUND: The epigenome represents an important target of environmental pollution. Early-life exposure to polychlorinated biphenyls (PCBs) modifies sex steroid enzymes and receptor transcription patterns. Steroid receptors, such as androgen receptor (AR), function as coregulators of histone modification enzymes. AIM: To clarify if a PCB early-life exposure might affect the epigenome in rat liver, we analyzed some histone post-translational modifications (H3K4me3 and H4K16Ac) and the corresponding histone remodeling enzymes, and the AR as a histone enzyme coregulator. RESULTS: We observed a decrease of H4K16Ac and H3K4me3 levels, possibly linked to the induction of chromatin-modifying enzymes SirtT1 and Jarid1b, and a decrease of AR. PCBs also seem to induce AR transcriptional activity. Some of the observed effects are sex dimorphic. CONCLUSION: Our data suggest that an early-life exposure to PCB sometimes modifies the epigenome in the offspring liver in a dimorphic way. AR might be involved in modulating PCB effects on the epigenome.

In Vitro Effects of PDGF Isoforms (AA, BB, AB and CC) on Migration and Proliferation of SaOS-2 Osteoblasts and on Migration of Human Osteoblasts.

PDGF is a major constituent of platelet rich plasma (PRP), responsible of chemotactic and possibly of mitogenic effects of PRP on osteoblasts. PDGF family includes 5 isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, all expressed in platelets except PDGF-DD. Aim of this study was to analyze the effect of recombinant hPDGF-A, -AB, -B and -C, on migration and proliferation of a human osteoblastic cell line, SaOS-2. Preliminary observations on cell migration were also done in primary cultures of human osteoblasts. In vitro microchemotaxis and (3)H-thymidine mitogenic assays were used. While PDGF-AB is active at concentrations present in PRP, PDGF-AA and BB are chemotactic only at much higher doses. PDGF-C is totally inactive alone or together with the active isoforms. PDGF-AA, PDGF-BB and PDGF-C stimulate SaOS-2 proliferation only at the highest dose tested, while PDGF-AB is ineffective. Primary osteoblasts are less sensitive than SaOS-2 and progressively lose responsiveness with increasing passages in culture, in line with loss of cell differentiation. The different PDGF isoforms act differentially on osteoblasts, the-AB isoform appearing the major responsible of the PRP chemiotaxis. PDGF, at the concentrations present in PRP, does not affect cell proliferation.

Sexual differentiation of the rodent hypothalamus: hormonal and environmental influences.

Brain sexual differentiation is a complex developmental phenomenon influenced by the genetic background, sex hormone secretions and environmental inputs, including pollution. The main hormonal drive to masculinize and defeminize the rodent brain is testosterone secreted by the testis. The hormone does not influence sex brain differentiation only in its native configuration, but it mostly needs local conversion into active metabolites (estradiol and DHT) through the action of specific enzymatic systems: the aromatase and 5alpha-reductase (5alpha-R), respectively. This allows the hormone to control target cell gene expression either through the estrogen (ER) or the androgen (AR) receptors. The developmental profile of testosterone metabolizing enzymes, different in the two sexes, is therefore of the utmost importance in affecting the bioavailability of the steroids active in brain differentiation. Widely diffused pollutants, like polychlorinated biphenyls (PCBs) are able to affect the production and/or action of testosterone metabolites, exerting detrimental influences on reproduction and sex behavior. The main studies performed in our and other laboratories concerning the pattern of expression and the control of the enzymatic systems involved in brain androgen action and metabolism are shortly reviewed. Some recent data on the influence exerted by PCBs on these metabolic systems are also reported.

Ontogenetic development, sexual differentiation, and effects of Aroclor 1254 exposure on expression of the arylhydrocarbon receptor and of the arylhydrocarbon receptor nuclear translocator in the rat hypothalamus.

Interaction of polychlorinated biphenyls (PCBs) with the aryl hydrocarbon receptor (AhR)/nuclear translocator (ARNT) system might interfere with the mechanisms controlling the sexual differentiation of the developing hypothalamus. The aim of this study was to evaluate the presence of AhR/ARNT in brain cells and the developmental profile of their expression in the hypothalamus of male and female rats during the perinatal period. Brain accumulation of the main PCB congeners after prenatal exposure to Aroclor 1254 and its influence on hypothalamic expression of AhR/ARNT was also assessed. The results show that: (a) AhR and ARNT are expressed both in neurons and in glia; (b) AhR expression progressively increases in the developing hypothalamus particularly in males, while ARNT is relatively constant in both sexes; (c) the prenatal administration of Aroclor to dams produces a differential accumulation of PCBs, depending on the chlorine atom number, and stimulates AhR expression only in the male hypothalamus. In conclusion, the developing male hypothalamus might be more sensitive to disrupting potential of PCBs.

Reflections on the diseases linked to mutations of the androgen receptor.

This review summarizes the most recent information on two pathologies linked to mutations of the androgen receptor, namely, the complete androgen insensitivity syndrome (CAIS) and the spinal and bulbar muscular atrophy (SBMA or Kennedy's disease). Data on the clinical manifestations of the two diseases are presented, together with the most relevant findings on their physiopathology and genetics.

Diferenciação sexual do cérebro: do animal experimental ao homem / Sexual differentiation of the brain: from the experimental animals to human beings

As diferenças existentes entre machos e fêmeas na morfologia e nas modalidades de funcionamento de numerosos núcleos cerebrais são determinadas, em grande parte, pelo ambiente hormonal presente durante o desenvolvimento embrionário. Os hormônios esteróides, em modo particular a testosterona, desenvolvem um papel crucial na masculinização dos centros cerebrais que governarão a secreção de vários hormônios hipotalâmicos e hipofisários, o comportamento sexual e a capacidade de aprendizagem e de memorização do indivíduo adulto. Nos últimos anos, surgiram dois aspectos importantes em relação ao mecanismo de ação da testosterona: 1) sua conversão, diretamente em nível de células-alvo, em compostos em grau de amplificar ou diversificar sua ação; 2) a presença, no sistema nervoso central (em particular no hipotálamo), da 5-alfa-redutase e da aromatase, enzimas necessárias a tais transformações. A interação dos metabólitos ativos resultantes (respectivamente, diidrotestosterona e estradiol) com específicos receptores intracelulares induz a transcrição de genes que determinam a diferenciação, em sentido masculino, das estruturas cerebrais em via de desenvolvimento. Nesta breve revisão, descrevem-se os conhecimentos atuais das principais características das duas vias enzimáticas que medeiam efeitos de diferenciação dos androgênios sobre o cérebro embrionário. Particular atenção é direcionada à discussão dos dados experimentais, obtidos principalmente nos roedores, em relação aos efeitos dos estrogênios de origem androgênica sobre a diferenciação sexual do cérebro. Discute-se também o papel co-primário que o genótipo desenvolve na diferenciação dimórfica do cérebro. Por fim, aborda-se a possível influência dos metabólitos ativos da testosterona sobre a diferenciação sexual do cérebro humano à luz de algumas doenças que comportam desequilíbrio nos níveis normais ou no mecanismo de ação dos hormônios gonadais durante o desenvolvimento embrionário.