In two experiments, immature female Sprague-Dawley rats treated with superovulatory doses of pregnant mare serum gonadotropin (PMSG) were used to study the effects of antiandrogen, hydroxyflutamide, on steroid production, particularly the biologically active androgens, testosterone, 5α-dihydrotestosterone, and androstenedione. In the first experiment, the animals were given either 5 mg hydroxyflutamide or vehicle alone at 30 and 36 h following 40 IU PMSG. Compared to the vehicle group, hydroxyflutamide treatment significantly reduced the percentage of degenerate oocytes recovered from oviducts (p<0.05). Serum levels of aromatizable androgens, testosterone and androstenedione, and their aromatized product, estradiol-17ß significantly decreased (p<0.05) in hydroxyflutamide-treated group; however, the serum concentrations of nonaromatizable androgen, 5α-dihydrotestosterone, was not statistically different between the two groups. In the second experiment, ovaries stimulated with 4 or 40 IU PMSG were obtained 48 h later and cultured in the presence and absence of hydroxyflutamide (10⁻⁵M) and/or testosterone (10⁻⁷ M) to study [4⁻¹⁴C] pregnenolone metabolism to major steroids. In 40 IU stimulated ovaries, hydroxyflutamide significantly decreased the metabolism of pregnenolone to progesterone (p<0.01) and androstenedione (p<0.01) while the production of estradiol-17ß increased significantly (p<0.05); however, pregnenolone conversions to testosterone and 5α-dihydrotestosterone were not statistically different between the untreated and hydroxyflutamide-treated cultures. Testosterone completely reversed the hydroxyflutamide-induced alteration of pregnenolone metabolism. In contrast, there was no difference in the pregnenolone conversion patterns between the untreated and hydroxyflutamide or hydroxyflutamide plus testosterone groups in the culture of ovaries stimulated with 4 IU PMSG. Present results confirm previous reports that antiandrogen, hydroxyflutamide, decreases the percentage of abnormal oocytes recovered from superovulating rats, and indicates that this hydroxyflutamide effect may be partly mediated by altered ovarian steroidogenesis, specifically the reduced hypersecretion of aromatizable androgens, testosterone and androstenedione, and/or estradiol-17ß. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Thesis (Ph.D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 201-254) Also available in print.

L'exposition durant la vie périnatale à des perturbateurs endocriniens (PE) anti-androgéniques, induit des altérations durables du système reproducteur mâle. Si leur action a été objectivée chez l’homme et l’animal, la question du seuil de toxicité reste entière, car l’exposition environnementale correspond à de faibles doses. Ici, nous exposons in utero des rats mâles à de faibles doses de flutamide: 10 mg/kg/j ; 1; 3 doses faibles selon la définition de l'OMS 0.1; 0.01; 0.001 et 0. L'exposition fœtale induit chez l’adulte (1) des altérations morphologiques à la dose 10 mg/kg/j (diminution du poids des organes du tractus génital et une diminution du compte spermatique); (2) des altérations cellulaires à partir de 1mg (augmentation de l'apoptose des cellules germinales) et (3) des altérations moléculaires à toutes les doses testées pour MCL1, BCL2, XIAP, HSPA2, ELAVL1 et MOV10L1. Ces altérations s’intègrent dans la dérégulation de 2 voies de signalisation expliquant l’apoptose des cellules germinalesLorsque l’exposition a lieu à l’âge l’adulte dans les mêmes conditions, les effets morphologiques et cellulaires sont observés uniquement à 10mg, les effets moléculaires uniquement aux doses 10 et 1 mg. Ces effets ne sont plus observés 2 mois après l’arrêt de l’exposition. En conclusion : Nous avons (i) identifié des voies de signalisation impliquées dans le phénotype d’infertilité induite par une exposition à de faibles doses d’anti-androgène ; (ii) montré que ces voies étaient actives aux plus faibles doses testées prédisposant à une infertilité masculine programmée in utero / Exposure during perinatal life to anti-androgenic endocrine disruptors (ED) induces sustained alterations of the male reproductive system. Although their action has been objectified in humans and animals, the question of the threshold of toxicity remains unchanged, as environmental exposure corresponds to low doses. Here, we exposed male rats, in utero, to low doses of flutamide: 10 mg/kg/d; 1; 3 low doses as defined by WHO 0.1; 0.01; 0.001 and 0. Fetal exposure induced in adults (1) morphological alterations at a dose of 10 mg/kg/day (decrease in the weight of organs of the genital tract and a decrease in the sperm count); (2) cellular alterations from 1mg (increase in germ cell apoptosis) and (3) molecular alterations at all doses tested for MCL1, BCL2, XIAP, HSPA2,LAVL1 and MOV10L1. These alterations are integrated in the deregulation of 2 signaling pathways explaining the apoptosis of the germ cellsWhen exposure occurs at adult age under the same conditions, morphological and cellular effects are observed only at 10mg, molecular effects only at doses 10 and 1 mg. These effects are no longer observed 2 months after discontinuation of exposure. In conclusion: We have (i) identified signaling pathways involved in the phenotype of infertility induced by exposure to low doses of antiandrogen; (ii) showed that these pathways were active at the lowest doses tested predisposing to programmed male infertility in utero

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Les perturbateurs endocriniens (PE) font l’objet d’une controverse scientifique à l’heure actuelle car, pour certains scientifiques, ces molécules n’auraient pas de seuil de toxicité et agiraient à de très faibles concentrations. Cette particularité proviendrait du fait que les PE, quelles que soient leurs origines, agissent sur les hormones et leurs récepteurs. A ce titre, ils seraient assujettis aux mêmes règles que les hormones endogènes. Leurs effets à de très faibles doses s’expliqueraient par la sensibilité des récepteurs hormonaux aux faibles fluctuations de concentrations. L’absence de seuil d’effet pour les PE s’expliquerait par différents mécanismes, dont des différences d’affinité suivant la concentration entre substrat et récepteur, qui engendreraient des effets doses non monotones. Un certain nombre de revues remettent en cause ces hypothèses et les observations qui les accompagnent. Elles indiquent notamment le fait que ces données manquent de répétitions, qu’elles sont principalement issues d’études in vitro ou sur des modèles d’organismes en développement et non-nécessairement misent en perspective avec des effets toxiques pour l’organisme. Ainsi ces données ne seraient pas suffisantes pour statuer dans ce débat.Dans ce contexte, afin d’apporter de nouvelles données expérimentales au débat, nous avons cherché à caractériser les effets aux faibles doses d’un PE de référence, le flutamide (FLU), sur l’appareil reproducteur du rat mâle adulte. Pour ce faire, nous avons conduit trois études successives au cours desquelles nous avons exposé des rats Wistar de 7 semaines à une large gamme de doses de FLU. La dose la plus forte est connue pour induire des lésions testiculaires et une atrophie de la prostate. Les autres doses correspondent à la NOAEL définie dans une étude publiée précédemment, et à cette valeur divisée par 10, 100 et 1000, ce qui correspond à des faibles doses selon la définition de l’OMS. Pour chaque étude, chaque groupe était composé de 16 animaux gavés quotidiennement pendant 28 jours. Ces études ont permis d'explorer différents paramètres. Dans le cadre de la caractérisation du mode d'action du flutamide sur le testicule (Exposition --> blocage des récepteurs aux androgènes de l'hypophyse --> augmentation de la LH circulante --> prolifération des cellules de Leydig --> Hyperplasie), nous avons montré que, pour chaque évènement clé, l’effet dose est monotone et qu’il est possible de définir un seuil. Par ailleurs, nous proposons le Pdgfd, un facteur de croissance, comme médiateur de l’effet mitogène de la LH sur les cellules de Leydig.Pour la prostate, le mode d'action du flutamide n’étant pas complètement élucidé à ces niveaux de doses, le travail a été plus exploratoire. Afin d'expliquer, aux niveaux moléculaire et cellulaire, l'origine de la baisse du poids des prostates induites par le flutamide dès 1 mg/kg/j, nous avons exploré trois hypothèses: (1) l'augmentation de l'apoptose, avec un test d'activité des caspases 3 et 9 qui montre une activité supérieure pour le groupe forte dose de 10 mg/kg/j de flutamide, (2) la diminution de la prolifération, avec un marquage ki67 qui ne montre pas de différence entre le groupe témoin et la forte dose, et enfin (3) la baisse de la fonction de sécrétion de la prostate, évaluée par un western blot dirigé contre la probasine qui montre une baisse de sa production. Dans le même temps, nous avons montré, au niveau des transcrits hépatiques (RT-qPCR), que le flutamide exerce un effet-dose monotone sur certaines enzymes impliquées dans le métabolisme des xénobiotiques. En conclusion, dans ce modèle, pour l'ensemble des paramètres explorés, sur le testicule, l’hypophyse, la prostate, le sang et le foie, nous n'avons observé que des effets à seuil et aucun effet non monotone. / The dose-response characterization of Endocrine disrupting chemicals (EDCs) toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low concentration. A part of the scientific community, suggests these molecules have no toxicity threshold and act at very low concentrations. This assumption stems from the fact that EDCs, whatever their origins, act on hormones and their receptors. As such, they would be subject to the same rules as the endogenous hormones. Effects at very low doses could be explained by the sensitivity of the hormone receptor to small fluctuations in concentrations. The lack of threshold for EDCs may be explained by different mechanisms, including affinity differences according to concentrations between substrate and receptor, which would generate non-monotonic dose-responses. A number of reviews challenge this assumption and the linked observations. They argue that the data are missing replication; they are mainly from in vitro studies or conducted in developing models and not-necessarily associated with toxic effects on the organism. Thus, these data are not sufficient to rule in this debate.In this context, in order to provide new experimental data to this debate, we worked to characterize the effects at low doses of a reference EDCs, flutamide (FLU) on the reproductive system of adult male rats. In that aim, we conducted three successive studies exposing Wistar rats of 7 weeks at a wide range of Flu doses. The highest dose is known to induce testicular damage and atrophy of the prostate gland. Other doses correspond to the defined NOAEL in a study published earlier, and to this value divided by 10, 100 and 1000, corresponding to low doses according to the WHO definition. For each study, each group consisted of 16 animals exposed daily by gavage for 28 days. During characterization of flutamide mode of action on the testis (Exposition -> androgen receptors blocking in the pituitary gland -> increase in circulating LH -> Leydig cells proliferation -> Leydig cells Hyperplasia), we have shown that for every key event, dose-response curve is monotonous and we were able to define a threshold. Furthermore, we suggest that the Pdgfd, a growth factor, mediates the mitogenic effect of LH on Leydig cells.In the prostate gland, the mode of action of flutamide is not completely elucidated at these dose levels, so the work was more exploratory. To explain, at the molecular and cellular levels, the origin of prostate atrophy induced by flutamide from 1 mg/kg/day, we explored three hypotheses: (1) the increase in apoptosis, with a caspases 3 and 9 activity test, which shows a higher activity for the high dosage group of 10 mg/kg/day of flutamide, (2) the decreasing of epithelium proliferation, with Ki67 marker which shows no difference between the control group and the high dose, and finally (3) the decrease in prostate secretory function, assessed by western blot against the Probasin which shows a drop in production.At the same time, we have shown in liver that flutamide has an effect on enzymes involved in the metabolism of xenobiotics with monotonic dose-response.In conclusion, in this model, for all investigated parameters on the testis, pituitary, prostate, blood and liver, we only observed threshold effects and no non-monotonic dose-responses.

Introdução: Com comportamento biológico heterogêneo e alta incidência, o Câncer de Próstata (CaP), apresenta uma grande variabilidade na agressividade da neoplasia. O CaP primário possuiu altas taxas de cura quando diagnosticado na sua forma inicial. Aproximadamente 35% dos pacientes acometidos pela neoplasia evoluem para a doença metastática. A aquisição de um fenótipo independente de andrógeno pelas células cancerosas da próstata é a alteração que apresenta o pior prognóstico para os pacientes. Uma investigação a respeito dos mecanismos de resistência ao tratamento hormonal bem como sobre possíveis mecanismos proporcionados por moléculas reguladoras, como os microRNAs, podem nos auxiliar na compreensão desse fenótipo resistente. Objetivos: Avaliar o perfil de expressão dos miR-221, miR-222, miR-23b e miR-27b e dos genes p27 Kip-1, CCNG1 e RA em amostras teciduais de pacientes com CaP primário que responderam ou não a terapia hormonal e correlacionar com os fatores prognósticos clássicos do CaP, além de avaliar o papel dos microRNAs anti-miR-221, anti-miR-222, miR-23 e miR-27b em experimentos in vitro utilizando ensaios de apoptose, ciclo e proliferação celular em ambientes exposto e não exposto a Flutamida (FLU) em linhagem celular sabidamente resistente à castração (PC-3). Materiais e Métodos: Selecionamos retrospectivamente 44 amostras de tumores primários de pacientes com CaP clinicamente localizado e que foram submetidos à prostatectomia radical. Dos 44 pacientes apenas nove continuaram a responder a hormônio terapia e 35 se mostraram resistente em menos de um ano de tratamento. O grupo controle foi constituído de dez espécimes cirúrgicos de pacientes que possuíam Hiperplasia Prostática Benigna (HPB). Para análise de expressão gênica e dos microRNAs os materiais genéticos foram obtidos utilizando protocolos convencionais de extração e técnica de qRT-PCR utilizando primers específicos para cada tipo de microRNA e gene. Os ensaios celulares de avaliação da apoptose, ciclo e proliferação celular foram realizados no citômetro MUSE utilizando linhagem celular PC-3 seguindo as recomendações do fabricante. Os grupos nos três ensaios eram constituídos das seguintes formas: controle, microRNA, FLU e microRNA+FLU. Resultados: Encontramos subexpressão dos miR-221, miR-222, miR- 23b e miR-27b no CaP em comparação com o HPB, em relação aos genes, foi observada superexpressão dos três genes analisados, p27 Kip-1, CCNG1 e RA. Encontramos correlação significativa entre a maior expressão do miR-23b e o grupo de pacientes que possuíam PSA > 10ng/mL. No ensaio de apoptose encontramos relações significativas entre as taxas de apoptose total nos grupos tratados com miR-23b+FLU e miR-27b+FLU em relação ao controle (p=0,0006 e p=0,003 respectivamente). No ensaio de ciclo celular, demonstramos que dentre os grupos já mencionados anteriormente, o que melhor demonstrou resultado foi o grupo tratado apenas com FLU, reduzindo o número de células na fase final, G2-M (p > 0,0001). Igualmente ao ciclo celular, o ensaio de proliferação demonstrou melhores resultados quando as células foram tratadas com FLU, ocorrendo uma diminuição discreta quando comparadas ao grupo controle (p < 0,05). Conclusão: Nossos resultados demonstram subexpressão dos 4 microRNAs estudados nas amostras dos pacientes com CaP e superexpressão dos três genes analisados nestes mesmos casos. Postulamos que exista um efeito sinérgico entre os microRNAs 23b/27b juntamente com a FLU no ensaio de apoptose, e demonstramos que apenas a FLU obteve resultado significativo no controle do ciclo celular e de células em proliferação em linhagem PC-3 / Introduction: Prostate Cancer (PCa) is a heterogeneous disease heterogeneous with high incidence, presents a great variability in the aggressiveness of the neoplasia. Primary PCa has high cure rates when diagnosed in its initial form. Approximately 35% of the patients affected by the neoplasia evolve to metastatic disease. The acquisition of an androgenindependent phenotype by prostate cancer cells is the change that presents the worst prognosis for patients. An investigation about mechanisms of resistance to hormone treatment as well as possible mechanisms provided by regulatory molecules, such as microRNAs, may help us to understand this resistant phenotype. Objectives: To evaluate the expression profile of miR-221, miR-222, miR-23b and miR-27b and p27 Kip-1, CCNG1 and AR genes in tissue samples from patients with primary PCa who have or have not responded to hormone therapy and to evaluate the role of anti-miR-221, antimiR- 222, miR-23 and miR-27b microRNAs in in vitro experiments using apoptosis, cycling and cell proliferation assays in environments exposed and not exposed to Flutamide (FLU) in cell line known to be resistant to castration (PC-3). Materials and Methods: We retrospectively selected 44 primary tumor samples from patients with clinically localized PCa and who underwent radical prostatectomy. Of the 44 patients, only nine continued to respond to hormone therapy and 35 were resistant in less than one year of treatment. The control group consisted of ten surgical specimens from patients who had Benign Prostatic Hyperplasia (BPH). For gene expression analysis and microRNAs the genetic materials were obtained using conventional extraction protocols and qRT-PCR technique using primers specific for each type of microRNA and gene. Cell assays of apoptosis, cycle and cell proliferation were performed on the MUSE cytometer using PC-3 cell line following the manufacturer\'s recommendations. The groups in the three trials were composed of the following forms: control, microRNA, FLU and microRNA+FLU. Results: We found subexpression of the miR-221, miR-222, miR-23b and miR-27b in PCa compared to BPH, in relation to the genes, overexpression of the three genes analyzed, p27 Kip-1, CCNG1 and AR was observed.We found a significant correlation between the greater expression of miR-23b and the group of patients who had PSA > 10ng/mL. In the apoptosis assay we found significant relationships between total apoptosis rates in the groups treated with miR-23b+FLU and miR-27b+FLU in relation to the control (p=0.0006 and p=0.003 respectively). Similarly to the cell cycle, the proliferation assay showed better results when the cells were treated with FLU, in this case a discrete decrease occurring when compared to the control group (p < 0.05). Conclusion: Our results demonstrate subexpression of the 4 microRNAs studied in the samples of patients with PCa. An anarchic expression of the p27 Kip-1 gene, overexpression of CCNG1 and AR genes in cases. We postulated that there is a synergistic effect between -23b/-27b microRNAs along with FLU in the apoptosis assay, and we demonstrated that only FLU had a significant effect on cell cycle control and proliferating in PC-3 cell line

Le cancer de la prostate est un problème de société majeur équivalent au cancer du sein chez la femme. La plupart de ces cancers sont hormono-dépendants et un des traitements utilisés est l'administration d'anti-androgènes non stéroïdiens. Ce sont des molécules qui inhibent de manière compétitive les effets des androgènes en se fixant sur le site de liaison du récepteur AR. Les anti-androgènes non stéroïdiens commerciaux sont la flutamide et son métabolite actif, le 2-hydroxyflutamide, la nilutamide et la bicalutamide. Néanmoins, à cause des phénomènes de résistance aux médicaments qui se manifestent après un traitement de longue durée, il devient impératif de concevoir de nouvelles molécules plus efficaces. Nous avons choisi d'incorporer un groupe ferrocène au niveau de la structure des anti-androgènes, car il possède des propriétés cytotoxiques intéressantes. Ce travail de recherche présente donc la synthèse d'analogues d'anti-androgènes non stéroïdiens commerciaux couplés avec des groupements ferrocène. Une fois synthétisés, ils sont soumis à une série de tests biologiques entrepris au sein du laboratoire. Ils comprennent, d'une part, des tests d'affinité relative de liaison (RBA) au récepteur à androgènes (AR) et, d'autre part, une série de tests anti-prolifératifs réalisés in vitro sur des lignées cellulaires tumorales prostatiques. Ces tests nous permettront de définir si ces molécules possèdent un intérêt particulier dans le cadre de la lutte contre le cancer de la prostate hormono-dépendant ou indépendant.

Introdução: Com comportamento biológico heterogêneo e alta incidência, o Câncer de Próstata (CaP), apresenta uma grande variabilidade na agressividade da neoplasia. O CaP primário possuiu altas taxas de cura quando diagnosticado na sua forma inicial. Aproximadamente 35% dos pacientes acometidos pela neoplasia evoluem para a doença metastática. A aquisição de um fenótipo independente de andrógeno pelas células cancerosas da próstata é a alteração que apresenta o pior prognóstico para os pacientes. Uma investigação a respeito dos mecanismos de resistência ao tratamento hormonal bem como sobre possíveis mecanismos proporcionados por moléculas reguladoras, como os microRNAs, podem nos auxiliar na compreensão desse fenótipo resistente. Objetivos: Avaliar o perfil de expressão dos miR-221, miR-222, miR-23b e miR-27b e dos genes p27 Kip-1, CCNG1 e RA em amostras teciduais de pacientes com CaP primário que responderam ou não a terapia hormonal e correlacionar com os fatores prognósticos clássicos do CaP, além de avaliar o papel dos microRNAs anti-miR-221, anti-miR-222, miR-23 e miR-27b em experimentos in vitro utilizando ensaios de apoptose, ciclo e proliferação celular em ambientes exposto e não exposto a Flutamida (FLU) em linhagem celular sabidamente resistente à castração (PC-3). Materiais e Métodos: Selecionamos retrospectivamente 44 amostras de tumores primários de pacientes com CaP clinicamente localizado e que foram submetidos à prostatectomia radical. Dos 44 pacientes apenas nove continuaram a responder a hormônio terapia e 35 se mostraram resistente em menos de um ano de tratamento. O grupo controle foi constituído de dez espécimes cirúrgicos de pacientes que possuíam Hiperplasia Prostática Benigna (HPB). Para análise de expressão gênica e dos microRNAs os materiais genéticos foram obtidos utilizando protocolos convencionais de extração e técnica de qRT-PCR utilizando primers específicos para cada tipo de microRNA e gene. Os ensaios celulares de avaliação da apoptose, ciclo e proliferação celular foram realizados no citômetro MUSE utilizando linhagem celular PC-3 seguindo as recomendações do fabricante. Os grupos nos três ensaios eram constituídos das seguintes formas: controle, microRNA, FLU e microRNA+FLU. Resultados: Encontramos subexpressão dos miR-221, miR-222, miR- 23b e miR-27b no CaP em comparação com o HPB, em relação aos genes, foi observada superexpressão dos três genes analisados, p27 Kip-1, CCNG1 e RA. Encontramos correlação significativa entre a maior expressão do miR-23b e o grupo de pacientes que possuíam PSA > 10ng/mL. No ensaio de apoptose encontramos relações significativas entre as taxas de apoptose total nos grupos tratados com miR-23b+FLU e miR-27b+FLU em relação ao controle (p=0,0006 e p=0,003 respectivamente). No ensaio de ciclo celular, demonstramos que dentre os grupos já mencionados anteriormente, o que melhor demonstrou resultado foi o grupo tratado apenas com FLU, reduzindo o número de células na fase final, G2-M (p > 0,0001). Igualmente ao ciclo celular, o ensaio de proliferação demonstrou melhores resultados quando as células foram tratadas com FLU, ocorrendo uma diminuição discreta quando comparadas ao grupo controle (p < 0,05). Conclusão: Nossos resultados demonstram subexpressão dos 4 microRNAs estudados nas amostras dos pacientes com CaP e superexpressão dos três genes analisados nestes mesmos casos. Postulamos que exista um efeito sinérgico entre os microRNAs 23b/27b juntamente com a FLU no ensaio de apoptose, e demonstramos que apenas a FLU obteve resultado significativo no controle do ciclo celular e de células em proliferação em linhagem PC-3 / Introduction: Prostate Cancer (PCa) is a heterogeneous disease heterogeneous with high incidence, presents a great variability in the aggressiveness of the neoplasia. Primary PCa has high cure rates when diagnosed in its initial form. Approximately 35% of the patients affected by the neoplasia evolve to metastatic disease. The acquisition of an androgenindependent phenotype by prostate cancer cells is the change that presents the worst prognosis for patients. An investigation about mechanisms of resistance to hormone treatment as well as possible mechanisms provided by regulatory molecules, such as microRNAs, may help us to understand this resistant phenotype. Objectives: To evaluate the expression profile of miR-221, miR-222, miR-23b and miR-27b and p27 Kip-1, CCNG1 and AR genes in tissue samples from patients with primary PCa who have or have not responded to hormone therapy and to evaluate the role of anti-miR-221, antimiR- 222, miR-23 and miR-27b microRNAs in in vitro experiments using apoptosis, cycling and cell proliferation assays in environments exposed and not exposed to Flutamide (FLU) in cell line known to be resistant to castration (PC-3). Materials and Methods: We retrospectively selected 44 primary tumor samples from patients with clinically localized PCa and who underwent radical prostatectomy. Of the 44 patients, only nine continued to respond to hormone therapy and 35 were resistant in less than one year of treatment. The control group consisted of ten surgical specimens from patients who had Benign Prostatic Hyperplasia (BPH). For gene expression analysis and microRNAs the genetic materials were obtained using conventional extraction protocols and qRT-PCR technique using primers specific for each type of microRNA and gene. Cell assays of apoptosis, cycle and cell proliferation were performed on the MUSE cytometer using PC-3 cell line following the manufacturer\'s recommendations. The groups in the three trials were composed of the following forms: control, microRNA, FLU and microRNA+FLU. Results: We found subexpression of the miR-221, miR-222, miR-23b and miR-27b in PCa compared to BPH, in relation to the genes, overexpression of the three genes analyzed, p27 Kip-1, CCNG1 and AR was observed.We found a significant correlation between the greater expression of miR-23b and the group of patients who had PSA > 10ng/mL. In the apoptosis assay we found significant relationships between total apoptosis rates in the groups treated with miR-23b+FLU and miR-27b+FLU in relation to the control (p=0.0006 and p=0.003 respectively). Similarly to the cell cycle, the proliferation assay showed better results when the cells were treated with FLU, in this case a discrete decrease occurring when compared to the control group (p < 0.05). Conclusion: Our results demonstrate subexpression of the 4 microRNAs studied in the samples of patients with PCa. An anarchic expression of the p27 Kip-1 gene, overexpression of CCNG1 and AR genes in cases. We postulated that there is a synergistic effect between -23b/-27b microRNAs along with FLU in the apoptosis assay, and we demonstrated that only FLU had a significant effect on cell cycle control and proliferating in PC-3 cell line

The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and member of the basic helix-loop-helix Per/ARNT/Sim (bHLH/PAS) family of chemosensors and developmental regulators. As a member of the PAS domain family of transcription factors responsive to exogenous signals, the AhR exerts influence on many processes relating to cellular fate. The activation of AhR is widely associated with toxic endpoints related to dioxin exposure. However, the AhR also activates endogenous gene programs related to development, cellular growth, and differentiation. The AhR is able to bind a variety of ligands, leading to a wide range of biological outcomes. Recent reports have shown that the AhR can mediate tumor suppressive effects. As a ligand-activated transcription factor, the AhR has the potential to actuate a variety of transcriptional programs that are dependent on the AhR ligand. Our central hypothesis is that AhR ligands can be identified that are capable of initiating tumor suppressive functions of the AhR. We utilized complementary cell-based and in silico virtual screening approaches to identify potential AhR ligands. We developed homology models of the AhR ligand-binding domain (LBD) for virtual ligand screening (VLS) of small molecule libraries. This led to the identification of new AhR ligands 5,7- dihydroxyflavanone!and 5-hydroxy-7-methoxyflavone. Additional small molecule libraries were screened in parallel that led to identification of flutamide as a putative AhR ligand. Flutamide is clinically approved for the treatment of prostate cancer due to its ability to antagonize androgen receptor mediated transcription. We investigated the biological effects of flutamide in AhR positive cancer cells that do not express the androgen receptor and found that flutamide inhibited the growth of HepG2 cells. Suppression of AhR expression reversed the anti-proliferative effects of flutamide. We tested 15 structural analogs of flutamide, including the flutamide metabolite 2-hydroxyflutamide for activation of AhR transcriptional activity. Flutamide is unique in its ability to activate the AhR, and suppresses hepatoma cell growth. These data suggests that flutamide-induced AhR transcriptional activity is required to initiate the tumor suppressive effects. We examined changes in cell cycle checkpoint proteins after flutamide treatment and discovered increased expression of cell cycle inhibitory proteins p27[superscript Kip1] and p15[superscript INK]. We also found that transforming Growth Factor β1 (TGFβ1), which regulates both p27[superscript Kip1] and p15[superscript INK], is upregulated by flutamide. We demonstrate that TGFβ1 is upregulated by flutamide in an AhR-dependent manner and is required for suppression of proliferation by flutamide. We identify specific and unique transcriptional signatures of the AhR upon activation by flutamide, that are distinct from the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In summary, we characterize flutamide as an AhR ligand and demonstrate its AhR-dependent tumor suppressive effects in hepatoma cells. We provide the first direct evidence that AhR regulates TGFβ signaling in a ligand dependent manner. We demonstrate that the AhR-induced downstream transcriptional signature and subsequent biological effects are specific to the AhR ligand. Our studies have broad impact for characterizing the AhR as a new therapeutic target in hepatocellular carcinoma. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from March 28, 2013 - March 28, 2015

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / nÃo hà / A Flutamida à um medicamento anti-androgÃnico nÃo esterÃide aprovado para o tratamento do cÃncer de prÃstata. Seu efeito hepatotÃxico foi conhecido desde o inÃcio do perÃodo de comercializaÃÃo, hà mais de 10 anos. Em marÃo e junho de 2002, a Unidade de FarmacovigilÃncia da ANVISA recebeu as primeiras comunicaÃÃes de Ãbitos por hepatite fulminante associados ao uso da substÃncia (medicamento manipulado) em jovens do sexo feminino. A literatura mundial nunca registrara caso semelhante. O objetivo deste trabalho foi revisar e acrescentar informaÃÃes a respeito da seguranÃa da Flutamida quando utilizadas por pacientes do sexo feminino para o tratamento de hirsutismo, acne e alopecia androgenÃtica. Foram realizadas pesquisas para conhecer a indicaÃÃo aprovada em outros paÃses e as notificaÃÃes de reaÃÃes adversas graves em mulheres no banco de dados da OMS. AlÃm disso, por meio de busca ativa, tentou-se identificar outros casos no Brasil; rever as informaÃÃes das bulas de todos os medicamentos comercializados e analisar se as informaÃÃes obrigatÃrias e relevantes para diminuir o risco de reaÃÃes hepÃticas graves estavam presentes. Por fim, foi realizada pesquisa com os prescritores, por meio de questionÃrio eletrÃnico, para conhecer as informaÃÃes de que dispunham sobre a utilizaÃÃo do medicamento em mulheres e comparar estas informaÃÃes com uma revisÃo crÃtica da literatura no que tange à seguranÃa e eficÃcia. Os resultados de todas estas estratÃgias mostraram que a substÃncia tambÃm à utilizada off label em mulheres em outros paÃses, e tambÃm causou reaÃÃes adversas graves, mas nenhum Ãbito no sexo feminino fora relatado. Durante o trabalho, outros casos de Ãbito no Brasil foram identificados. Todas as bulas comercializadas no Brasil mencionavam que a substÃncia nÃo devia ser utilizada por mulheres, assim como mencionavam as reaÃÃes hepÃticas graves. As respostas dos prescritores ao questionÃrio permitem afirmar que a Flutamida se constitui em importante arsenal terapÃutico, e que o risco de seu uso vem sendo subestimado. O trabalho permite concluir que a Flutamida mostra-se eficaz para as condiÃÃes em que vem sendo utilizada em mulheres. Entretanto, os riscos evidenciam uma relaÃÃo benefÃcio/risco inaceitÃvel para as condiÃÃes estÃticas em que vem sendo empregada. / Flutamide is a non steroid anti-androgenic drug used to treat prostate cancer patients. When it became commercialized, more than 10 years ago, the hepatotoxic effect of flutamide was already recognized. In March and July 2002, the first cases of deaths in young females caused by fulminant hepatitis associated to off label use of this substance (magistral drug) was reported to the Pharmacovigilance Office/ANVISA. Up to now, similar cases were not described in the international literature. This work aimed to review and add information about the adequate use of flutamide by female patients to treat hirsutism, acne and androgenetic alopecia. To identify the standard prescriptions and the reported serious reactions in females worldwide, an extensive research through data provided by OMS was conducted. Moreover, using active search we looked for new local cases and review the information from package insert of all commercialized drugs in Brazil to analyze if the required and relevant information warning patients against the serious hepatic risks were provided. Finally, a survey with the prescribers was carried on through the web to identify the available information about the use of this drug in females and to make comparisons with data from the literature about the safety and efficacy of the use of this substance. The results showed that this substance is also used off label by women from other countries, who also presented serious adverse reactions. However, it was not reported any case of female death. Over the study, new cases of obituary occurred in Brazil, despite all analyzed package insert explain that this substance should not be used by females and mention serious hepatic reactions. The answers of the prescribers to the survey indicated that flutamide is an important therapeutic arsenal and that the risk of the use of this substance seem to have been underestimated. We concluded that flutamide is effective for the women treatment which it has been used. However, the threats of this drug point to the unacceptable use of this drug when applied for the esthetic finality.