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BACKGROUND: Prostate cancer (PC) remains a leading cause of cancer mortality and the most successful chemopreventative and treatment strategies for PC come from targeting the androgen receptor (AR). Although AR plays a key role, it is likely that other molecular pathways also contribute to PC, making it essential to identify and develop drugs against novel targets. Recent studies have identified peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor that regulates fatty acid (FA) metabolism, as a novel target in PC, and suggest that inhibitors of PPARγ could be used to treat existing disease. We hypothesized that PPARγ acts through AR-dependent and independent mechanisms to control PC development and growth and that PPARγ inhibition is a viable PC treatment strategy. METHODS: Immunohistochemistry was used to determine expression of PPARү in a cohort of patients with PC. Standard molecular techniques were used to investigate the PPARү signaling in PC cells as well a xenograft mouse model to test PPARү inhibition in vivo. Kaplan-Meier curves were created using cBioportal. RESULTS: We confirmed the expression of PPARү in human PC. We then showed that small molecule inhibition of PPARγ decreases the growth of AR-positive and -negative PC cells in vitro and that T0070907, a potent PPARγ antagonist, significantly decreased the growth of human PC xenografts in nude mice. We found that PPARγ antagonists or small interfering RNA (siRNA) do not affect mitochondrial activity nor do they cause apoptosis; instead, they arrest the cell cycle. In AR-positive PC cells, antagonists and siRNAs reduce AR transcript and protein levels, which could contribute to growth inhibition. AR-independent effects on growth appear to be mediated by effects on FA metabolism as the specific FASN inhibitor, Fasnall, inhibited PC cell growth but did not have an additive effect when combined with PPARγ antagonists. Patients with increased PPARү target gene expression, but not alterations in PPARү itself, were found to have significantly worse overall survival. CONCLUSIONS: Having elucidated the direct cancer cell effects of PPARγ inhibition, our studies have helped to determine the role of PPARγ in PC growth, and support the hypothesis that PPARγ inhibition is an effective strategy for PC treatment.
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PPAR gama/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Animais , Benzamidas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ácidos Graxos/biossíntese , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , PPAR gama/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Piridinas/farmacologia , Receptores Androgênicos/genética , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. METHODS: Mice were castrated and implanted with capsules to achieve castrate, normal, or sub-physiological levels of T. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue. FACS was used to quantify the percentages of purported prostate stem and transit amplifying (TA) cells in mouse prostates. Prostate tissues were also stained for the presence of CD68+ cells and RNA was extracted from prostate tissue or specific cell populations to measure changes in transcript levels with low T treatment. RESULTS: Despite having significantly different levels of T and DHT in the serum, T and DHT concentrations in prostate tissue from different T treatment groups were similar. Low T treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining prostate androgen levels. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all T treatment groups, demonstrating that the mouse prostate can maintain functional levels of androgens despite low serum T levels. Low T increased the frequency of prostate stem and TA cells in adult prostate tissue and caused major transcriptional changes in those cells. Gene ontology analysis suggested that low T caused inflammatory responses and immunofluorescent staining indicated that low T treatment led to the increased presence of CD68+ macrophages in prostate tissue. CONCLUSIONS: Low T alters the AR signaling axis which likely leads to maintenance of functional levels of prostate androgens. Low T also induces quantitative and qualitative changes in prostate stem cells which appear to lead to inflammatory macrophage infiltration. These changes are proposed to lead to an aggressive phenotype once cancers develop and may contribute to the poor outcomes in men with low T. Prostate 77:530-541, 2017. © 2016 Wiley Periodicals, Inc.
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Próstata/citologia , Próstata/metabolismo , Células-Tronco/metabolismo , Testosterona/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Orquiectomia/métodos , Próstata/patologia , Células-Tronco/patologia , Testosterona/sangueRESUMO
INTRODUCTION: Androgens regulate a wide array of physiological processes, including male sexual development, bone and muscle growth, and behavior and cognition. Because androgens play a vital role in so many tissues, changes in androgen signaling are associated with a plethora of diseases. How such varied responses are achieved by a single stimulus is not well understood. Androgens act primarily through the androgen receptor (AR), a hormone nuclear receptor that is expressed in a select variety of tissues. METHODS: In order to gain a better understanding of how the tissue-selective effects of androgens are achieved, we performed a comparison of microarray data, using previously published datasets and several of our own microarray datasets. These datasets were derived from clinically relevant, AR-expressing tissues dissected from rodents treated with the full androgen dihydrotestosterone (DHT). RESULTS: We found that there is a diverse response to DHT, with very little overlap of androgen regulated genes in each tissue. Gene ontology analyses also indicated that, while several tissues regulate similar biological processes in response to DHT, most androgen regulated processes are specific to one or a few tissues. Thus, it appears that the disparate physiological effects mediated by androgens begin with widely varying effects on gene expression in different androgen-sensitive tissues. CONCLUSION: The analysis completed in this study will lead to an improved understanding of how androgens mediate diverse, tissue-specific processes and better ways to assess the tissue-selective effects of AR modulators during drug development.
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Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Castração , Feminino , Perfilação da Expressão Gênica , Masculino , Ovário/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Próstata/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Pele/efeitos dos fármacosRESUMO
INTRODUCTION: Combined therapy with deferoxamine (DFO) and deferasirox (DFX) may be performed empirically when DFX monotherapy fails. Given the lack of published data on this therapy, the study goal was to assess the safety and efficacy of combined DFO/DFX therapy in a gerbil model. METHODS: Thirty-two female Mongolian gerbils 8-10 weeks old were divided into 4 groups (sham chelated, DFO, DFX, DFO/DFX). Each received 10 weekly injections of 200 mg/kg iron dextran prior to initiation of 12 weeks of chelation. Experimental endpoints were heart and liver weights, iron concentration and histology. RESULTS: In the heart, there was no significant difference among the treatment groups for wet-to-dry ratio, iron concentration and iron content. DFX-treated animals exhibited lower organ weights relative to sham-chelated animals (less iron-mediated hypertrophy). DFO-treated organs did not differ from sham-chelated organs in any aspects. DFX significantly cleared hepatic iron. No additive effects were observed in the organs of DFO/DFX-treated animals. CONCLUSIONS: Combined DFO/DFX therapy produced no detectable additive effect above DFX monotherapy in either the liver or heart, suggesting competition with spontaneous iron elimination mechanisms for chelatable iron. Combined therapy was well tolerated, but its efficacy could not be proven due to limitations in the animal model.
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Benzoatos/administração & dosagem , Desferroxamina/administração & dosagem , Sobrecarga de Ferro/tratamento farmacológico , Triazóis/administração & dosagem , Animais , Deferasirox , Modelos Animais de Doenças , Quimioterapia Combinada , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Gerbillinae , Coração , Fígado , Tamanho do Órgão , Resultado do TratamentoRESUMO
OBJECTIVE: Despite the availability of deferoxamine chelation therapy for more than 20 years, iron cardiomyopathy remains the leading cause of death in thalassemia major patients. Effective chelation of cardiac iron is difficult; cardiac iron stores respond more slowly to chelation therapy and require a constant gradient of labile iron species between serum and myocytes. We have previously demonstrated the efficacy of once-daily deferasirox in removing previously stored cardiac iron in the gerbil, but changes in cardiac iron were relatively modest compared with hepatic iron. We postulated that daily divided dosing, by sustaining a longer labile iron gradient from myocytes to serum, would produce better cardiac iron chelation than a comparable daily dose. METHODS: Twenty-four 8- to 10-week-old female gerbils underwent iron dextran-loading for 10 weeks, followed by a 1-week iron equilibration period. Animals were divided into three treatment groups of eight animals each and were treated with deferasirox 100 mg/kg/day as a single dose, deferasirox 100 mg/kg/day daily divided dose, or sham chelation for a total of 12 weeks. Following euthanasia, organs were harvested for quantitative iron and tissue histology. RESULTS: Hepatic and cardiac iron contents were not statistically different between the daily single-dose and daily divided-dose groups. However, the ratio of cardiac to hepatic iron content was lower in the divided-dose group (0.78% vs 1.11%, p = 0.0007). CONCLUSION: Daily divided dosing of deferasirox changes the relative cardiac and liver iron chelation profile compared with daily single dosing, trading improvements in cardiac iron elimination for less-effective hepatic chelation.
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Benzoatos/administração & dosagem , Cardiomiopatias/tratamento farmacológico , Modelos Animais de Doenças , Quelantes de Ferro/administração & dosagem , Sobrecarga de Ferro/tratamento farmacológico , Triazóis/administração & dosagem , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Deferasirox , Esquema de Medicação , Feminino , Gerbillinae , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Fígado/metabolismo , Fígado/patologia , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
INTRODUCTION: Iron cardiomyopathy is a lethal complication of transfusion therapy in thalassemia major. Nutritional supplements decreasing cardiac iron uptake or toxicity would have clinical significance. Murine studies suggest taurine may prevent oxidative damage and inhibit Ca2+-channel-mediated iron transport. We hypothesized that taurine supplementation would decrease cardiac iron-overloaded toxicity by decreasing cardiac iron. Vitamin E and selenium served as antioxidant control. METHODS: Animals were divided into control, iron, taurine, and vitamin E/selenium groups. Following sacrifice, iron and selenium measurements, histology, and biochemical analyses were performed. RESULTS: No significant differences were found in heart and liver iron content between treatment groups, except for higher hepatic dry-weight iron concentrations in taurine-treated animals (p < 0.03). Serum iron increased with iron loading (751 +/- 66 vs. 251 +/- 54 microg/dl, p < 0.001) and with taurine (903 +/- 136 microg/dl, p = 0.03). CONCLUSION: Consistent with oxidative stress, iron overload increased cardiac malondialdehyde levels, decreased heart glutathione peroxidase (GPx) activity, and increased serum aspartate aminotransferase. Taurine ameliorated these changes, but only significantly for liver GPx activity. Selenium and vitamin E supplementation did not improve oxidative markers and worsened cardiac GPx activity. These results suggest that taurine acts primarily as an antioxidant rather than inhibiting iron uptake. Future studies should illuminate the complexity of these results.
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Antioxidantes/uso terapêutico , Cardiomiopatias/prevenção & controle , Sobrecarga de Ferro/tratamento farmacológico , Miocárdio/metabolismo , Taurina/uso terapêutico , Alanina Transaminase/sangue , Animais , Antioxidantes/administração & dosagem , Aspartato Aminotransferases/sangue , Cardiomiopatias/etiologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Gerbillinae , Glutationa Peroxidase/análise , Coração/efeitos dos fármacos , Sobrecarga de Ferro/complicações , Complexo Ferro-Dextran/farmacocinética , Complexo Ferro-Dextran/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/química , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Malondialdeído/análise , Modelos Animais , Miocárdio/química , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo , Ácido Selênico , Compostos de Selênio/administração & dosagem , Compostos de Selênio/uso terapêutico , Taurina/administração & dosagem , Vitamina E/administração & dosagem , Vitamina E/uso terapêuticoRESUMO
AIM: To identify neuron-selective androgen receptor (AR) signaling inhibitors, which could be useful in the treatment of spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, a neuromuscular disorder in which deterioration of motor neurons leads to progressive muscle weakness. METHODS: Cell lines representing prostate, kidney, neuron, adipose, and muscle tissue were developed that stably expressed the CFP-AR-YFP FRET reporter. We used these cells to screen a library of small molecules for cell type-selective AR inhibitors. Secondary screening in luciferase assays was used to identify the best cell-type specific AR inhibitors. The mechanism of action of a neuron-selective AR inhibitor was examined in vitro using luciferase reporter assays, immunofluorescence microscopy, and immunoprecipitations. Rats were treated with the most potent compound and tissue-selective AR inhibition was examined using RT-qPCR of AR-regulated genes and immunohistochemistry. RESULTS: We identified the thiazole class of antibiotics as compounds able to inhibit AR signaling in a neuronal cell line but not a muscle cell line. One of these antibiotics, thiostrepton is able to inhibit the activity of both wild type and polyglutamine expanded AR in neuronal GT1-7 cells with nanomolar potency. The thiazole antibiotics are known to inhibit FOXM1 activity and accordingly, a novel FOXM1 inhibitor FDI-6 also inhibited AR activity in a neuron-selective fashion. The selective inhibition of AR is likely indirect as the varied structures of these compounds would not suggest that they are competitive antagonists. Indeed, we found that FOXM1 expression correlates with cell-type selectivity, FOXM1 co-localizes with AR in the nucleus, and that shRNA-mediated knock down of FOXM1 reduces AR activity and thiostrepton sensitivity in a neuronal cell line. Thiostrepton treatment reduces FOXM1 levels and the nuclear localization of beta-catenin, a known co-activator of both FOXM1 and AR, and reduces the association between beta-catenin and AR. Treatment of rats with thiostrepton demonstrated AR signaling inhibition in neurons, but not muscles. CONCLUSION: Our results suggest that thiazole antibiotics, or other inhibitors of the AR-FOXM1 axis, can inhibit AR signaling selectively in motor neurons and may be useful in the treatment or prevention of SBMA symptoms.
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Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Varfarina/farmacologia , Animais , Anticoagulantes/farmacologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Espectrometria de Massas , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , PPAR gama/efeitos dos fármacos , PPAR gama/metabolismo , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Transfusional therapy for thalassemia major and sickle cell disease can lead to iron deposition and damage to the heart, liver, and endocrine organs. Iron causes the MRI parameters T1, T2, and T2* to shorten in these organs, which creates a potential mechanism for iron quantification. However, because of the danger and variability of cardiac biopsy, tissue validation of cardiac iron estimates by MRI has not been performed. In this study, we demonstrate that iron produces similar T1, T2, and T2* changes in the heart and liver using a gerbil iron-overload model. METHODS AND RESULTS: Twelve gerbils underwent iron dextran loading (200 mg . kg(-1) . wk(-1)) from 2 to 14 weeks; 5 age-matched controls were studied as well. Animals had in vivo assessment of cardiac T2* and hepatic T2 and T2* and postmortem assessment of cardiac and hepatic T1 and T2. Relaxation measurements were performed in a clinical 1.5-T magnet and a 60-MHz nuclear magnetic resonance relaxometer. Cardiac and liver iron concentrations rose linearly with administered dose. Cardiac 1/T2*, 1/T2, and 1/T1 rose linearly with cardiac iron concentration. Liver 1/T2*, 1/T2, and 1/T1 also rose linearly, proportional to hepatic iron concentration. Liver and heart calibrations were similar on a dry-weight basis. CONCLUSIONS: MRI measurements of cardiac T2 and T2* can be used to quantify cardiac iron. The similarity of liver and cardiac iron calibration curves in the gerbil suggests that extrapolation of human liver calibration curves to heart may be a rational approximation in humans.
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Cardiomiopatias/metabolismo , Sobrecarga de Ferro/metabolismo , Ferro/análise , Imageamento por Ressonância Magnética , Miocárdio/química , Talassemia/terapia , Animais , Calibragem , Feminino , Gerbillinae , Humanos , Ferro/metabolismo , Fígado/química , Reprodutibilidade dos Testes , Especificidade da Espécie , Reação TransfusionalRESUMO
Iron cardiomyopathy remains the leading cause of death in patients with thalassemia major. Magnetic resonance imaging (MRI) is ideally suited for monitoring thalassemia patients because it can detect cardiac and liver iron burdens as well as accurately measure left ventricular dimensions and function. However, patients with thalassemia have unique physiology that alters their normative data. In this article, we review the physiology and pathophysiology of thalassemic heart disease as well as the use of MRI to monitor it. Despite regular transfusions, thalassemia major patients have larger ventricular volumes, higher cardiac outputs, and lower total vascular resistances than published data for healthy control subjects; these hemodynamic findings are consistent with chronic anemia. Cardiac iron overload increases the relative risk of further dilation, arrhythmias, and decreased systolic function. However, many patients are asymptomatic despite heavy cardiac burdens. We explore possible mechanisms behind cardiac iron-function relationships and relate these mechanisms to clinical observations.
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Cardiomiopatias/fisiopatologia , Sobrecarga de Ferro/fisiopatologia , Ferro/metabolismo , Talassemia/complicações , Soluções Tampão , Débito Cardíaco , Cardiomegalia/etiologia , Cardiomiopatias/diagnóstico , Cardiomiopatias/etiologia , Terapia por Quelação , Terapia Combinada , Coração/fisiopatologia , Humanos , Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/etiologia , Imageamento por Ressonância Magnética , Modelos Biológicos , Oxirredução , Volume Sistólico , Talassemia/tratamento farmacológico , Talassemia/metabolismo , Talassemia/terapia , Reação Transfusional , Resistência VascularRESUMO
Iron cardiomyopathy is the leading cause of death in transfusional iron overload, and men have twice the mortality of women. Because the prevalence of cardiac iron overload increases rapidly during the second decade of life, we postulated that there are steroid-dependent sex differences in cardiac iron uptake. To test this hypothesis, we manipulated sex steroids in mice with constitutive iron absorption (homozygous hemojuvelin knockout); this model mimics the myocyte iron deposition observed in humans. At 4 weeks of age, female mice were ovariectomized (OVX) and male mice were castrated (OrchX). Female mice received an estrogen implant (OVX + E) or a cholesterol control (OVX), whereas male mice received an implant containing testosterone (OrchX + T), dihydrotestosterone (OrchX + DHT), estrogen (OrchX + E), or cholesterol (OrchX). All animals received a high-iron diet for 8 weeks. OrchX, OVX, and OVX + E mice all had similar cardiac iron loads. However, OrchX + E males had a significant increase in cardiac iron concentration compared with OrchX mice (P < 0.01), whereas the OrchX + T and OrchX + DHT groups only trended higher (P < 0.06 and P < 0.15, respectively). Hormone treatments did not impact liver iron concentration in either sex. When data were pooled across hormone therapies, liver iron concentration was 25% greater in males than females (P < 0.01). In summary, we found that estrogen increased cardiac iron loading in male mice, but not in females. Male mice loaded 25% more hepatic iron than female mice regardless of the hormone treatment.
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Modelos Animais de Doenças , Sobrecarga de Ferro/metabolismo , Miocárdio/metabolismo , Fatores Sexuais , Animais , Sequência de Bases , Primers do DNA , Feminino , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Pyrvinium pamoate (PP) is a potent noncompetitive inhibitor of the androgen receptor (AR). Using a novel method of target identification, we demonstrate that AR is a direct target of PP in prostate cancer cells. We demonstrate that PP inhibits AR activity via the highly conserved DNA binding domain (DBD), the only AR inhibitor that functions via this domain. Furthermore, computational modeling predicts that pyrvinium binds at the interface of the DBD dimer and the minor groove of the AR response element. Because PP acts through the DBD, PP is able to inhibit the constitutive activity of AR splice variants, which are thought to contribute to the growth of castration resistant prostate cancer (CRPC). PP also inhibits androgen-independent AR activation by HER2 kinase. The antiandrogen activity of pyrvinium manifests in the ability to inhibit the in vivo growth of CRPC xenografts that express AR splice variants. Interestingly, PP was most potent in cells with endogenous AR expression derived from prostate or bone. PP was able to inhibit several other hormone nuclear receptors (NRs) but not structurally unrelated transcription factors. PP inhibition of other NRs was similarly cell-type selective. Using dual-energy X-ray absorptiometry, we demonstrate that the cell-type specificity of PP manifests in tissue-selective inhibition of AR activity in mice, as PP decreases prostate weight and bone mineral density but does not affect lean body mass. Our results suggest that the noncompetitive AR inhibitor pyrvinium has significant potential to treat CRPC, including cancers driven by ligand-independent AR signaling.
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Antagonistas de Receptores de Andrógenos/farmacologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Compostos de Pirvínio/farmacologia , Receptores Androgênicos/metabolismo , Absorciometria de Fóton , Antagonistas de Receptores de Andrógenos/efeitos adversos , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/uso terapêutico , Animais , Densidade Óssea/efeitos dos fármacos , Linhagem Celular Tumoral , Biologia Computacional , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Modelos Biológicos , Simulação de Acoplamento Molecular , Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Compostos de Pirvínio/efeitos adversos , Compostos de Pirvínio/química , Compostos de Pirvínio/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prostate cancer (PC) is both an age- and an androgen-dependent disease. Paradoxically, systemic levels of androgens decline with age as the risk of PC rises. While there is no correlation between systemic androgen levels and the risk of PC, systemic androgen levels do not reflect the levels of androgens in prostate tissue. In metastatic PC, changes in the androgen biosynthesis pathway during hormone therapy result in increased levels of androgens in cancer tissue and contribute to continued androgen receptor (AR) signaling. It is possible that similar changes occur in normal prostate tissue as androgen levels decline with age and that this contributes to tumorigenesis. In the present study, we sought to determine whether the rat prostate is able to maintain functional levels of androgens despite low serum testosterone levels. Rats were castrated and implanted with capsules to achieve castrate, normal, sub-physiological, and supra-physiological levels of testosterone. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of testosterone and dihydrotestosterone (DHT) in the serum and prostate tissue. Quantitative RT-PCR was used to quantify the expression of genes involved in the androgen/AR signaling axis. Despite significantly different levels of testosterone and DHT being present in the serum, testosterone and DHT concentrations in prostate tissue from different testosterone-treatment groups were very similar. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all the testosterone-treatment groups, demonstrating that the rat prostate can maintain a functional level of androgens despite low serum testosterone levels. Low-testosterone treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining functional androgen levels.
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Próstata/metabolismo , Testosterona/metabolismo , Androgênios/sangue , Androgênios/metabolismo , Animais , Vias Biossintéticas/genética , Expressão Gênica , Masculino , Próstata/patologia , Ratos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Esteroides/biossíntese , Testosterona/sangueRESUMO
Iron chelation is essential to patients on chronic blood transfusions to prevent toxicity from iron overload and remove excess iron. Deferasirox (DFX) is the most commonly used iron chelator in the United States; however, some patients are relatively refractory to DFX therapy. We postulated that vitamin C supplementation would improve the availability of transfusional iron to DFX treatment by promoting iron's redox cycling, increasing its soluble ferrous form and promoting its release from reticuloendothelial cells. Osteogenic dystrophy rats (n = 54) were given iron dextran injections for 10 weeks. Cardiac and liver iron levels were measured after iron loading (n = 18), 12 weeks of sham chelation (n = 18), and 12 weeks of DFX chelation (n = 18) at 75 mg/kg/day. Ascorbate supplementation of 150 ppm, 900 ppm, and 2250 ppm was used in the chow to mimic a broad range of ascorbate status; plasma ascorbate levels were 5.4 ± 1.9, 8.2 ± 1.4, 23.6 ± 9.8 µM, respectively (p < 0.0001). The most severe ascorbate deficiency produced reticuloenthelial retention, lowering total hepatic iron by 29% at the end of iron loading (p < 0.05) and limiting iron redistribution from cardiac and hepatic macrophages during 12 weeks of sham chelation. Most importantly, ascorbate supplementation at 2250 ppm improved DFX efficiency, allowing DFX to remove 21% more hepatic iron than ascorbate supplementation with 900 ppm or 150 ppm (p < 0.05). We conclude that vitamin C status modulates the release of iron from the reticuloendothelial system and correlates positively with DFX chelation efficiency. Our findings suggest that ascorbate status should be probed in patients with unsatisfactory response to DFX.
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Deficiência de Ácido Ascórbico , Ácido Ascórbico/sangue , Benzoatos/farmacologia , Quelantes de Ferro/farmacologia , Sobrecarga de Ferro , Ferro/sangue , Sistema Fagocitário Mononuclear/metabolismo , Triazóis/farmacologia , Animais , Deficiência de Ácido Ascórbico/sangue , Deficiência de Ácido Ascórbico/tratamento farmacológico , Deferasirox , Cobaias , Humanos , Sobrecarga de Ferro/sangue , Sobrecarga de Ferro/tratamento farmacológico , Fígado/metabolismo , Fígado/patologia , Sistema Fagocitário Mononuclear/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos MutantesRESUMO
Iron cardiomyopathy in ß-thalassemia major patients is associated with a vitamin D deficiency. Stores of 25-OH-D3 are markedly reduced, whereas the active metabolite, 1-25-(OH)-D3, is normal or increased. Interestingly, the ratio of 25-OH-D3 to 1-25-(OH)-D3 (a surrogate for parathyroid hormone [PTH]) is the strongest predictor of cardiac iron. Increased PTH and 1-25-OH-D3 levels have been shown to up-regulate L-type voltage-gated calcium channels (LVGCC), the putative channel for cardiac iron uptake. Therefore, we postulate that a vitamin D deficiency increases cardiac iron by altering LVGCC regulation. Hemojuvelin knockout mice were calcitriol treated, PTH treated, vitamin D-depleted, or untreated. Half of the animals in each group received the Ca(2+)-channel blocker verapamil. Mn(2+) was infused to determine LVGCC activity. Hearts and livers were harvested for iron, calcium, and manganese measurements as well as histology. Cardiac iron did not differ among the treatment groups; however, liver iron was increased in vitamin D-depleted animals (P < 0.0003). Cardiac iron levels did not correlate with manganese uptake but were proportional to cardiac calcium levels (r(2) = 0.6; P < 0.0001). Verapamil treatment reduced both cardiac (P < 0.02) and hepatic (P < 0.003) iron levels significantly by 34% and 28%, respectively. The association between cardiac iron and calcium levels was maintained after verapamil treatment (r(2) = 0.3; P < 0.008). Vitamin D depletion is associated with an increase in liver, but not cardiac, iron accumulation. Cardiac iron uptake was strongly correlated with cardiac calcium stores and was significantly attenuated by verapamil, suggesting that cardiac calcium and iron are related.
Assuntos
Cálcio/metabolismo , Audição/efeitos dos fármacos , Sobrecarga de Ferro , Ferro/metabolismo , Miocárdio/metabolismo , Animais , Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Modelos Animais de Doenças , Humanos , Sobrecarga de Ferro/tratamento farmacológico , Camundongos , Camundongos Knockout , Verapamil/farmacologiaRESUMO
MRI is gaining increasing importance for the noninvasive quantification of organ iron burden. Since transverse relaxation rates depend on iron distribution as well as iron concentration, physiologic and pharmacologic processes that alter iron distribution could change MRI calibration curves. This article compares the effect of three iron chelators, deferoxamine, deferiprone, and deferasirox, on R1 and R2 calibration curves according to two iron loading and chelation strategies. Thirty-three Mongolian gerbils underwent iron loading (iron dextran 500 mg/kg/wk) for 3 weeks followed by 4 weeks of chelation. An additional 56 animals received less aggressive loading (200 mg/kg/week) for 10 weeks, followed by 12 weeks of chelation. R1 and R2 calibration curves were compared to results from 23 iron-loaded animals that had not received chelation. Acute iron loading and chelation-biased R1 and R2 from the unchelated reference calibration curves but chelator-specific changes were not observed, suggesting physiologic rather than pharmacologic differences in iron distribution. Long-term chelation deferiprone treatment increased liver R1 50% (P < 0.01), while long-term deferasirox lowered liver R2 30.9% (P < 0.0001). The relationship between R1 and R2 and organ iron concentration may depend on the acuity of iron loading and unloading as well as the iron chelator administered.
Assuntos
Ferro/metabolismo , Fígado/química , Imageamento por Ressonância Magnética , Miocárdio/química , Animais , Benzoatos/farmacologia , Deferasirox , Deferiprona , Desferroxamina/farmacologia , Gerbillinae , Quelantes de Ferro/farmacologia , Piridonas/farmacologia , Sideróforos/farmacologia , Distribuição Tecidual , Triazóis/farmacologiaRESUMO
The potential interplay of glial cells with T cells during viral induced inflammation was assessed by comparing major histocompatibility complex molecule upregulation and retention on astrocytes and microglia. Transgenic mice expressing green fluorescent protein under control of the astrocyte-specific glial fibrillary acidic protein promoter were infected with a neurotropic coronavirus to facilitate phenotypic characterization of astrocytes and microglia using flow cytometry. Astrocytes in the adult central nervous system up-regulated class I surface expression, albeit delayed compared with microglia. Class II was barely detectable on astrocytes, in contrast to potent up-regulation on microglia. Maximal MHC expression in both glial cell types correlated with IFN-gamma levels and lymphocyte accumulation. Despite a decline of IFN-gamma concomitant to virus clearance, MHC molecule expression on glia was sustained. These data demonstrate distinct regulation of both class I and class II expression by microglia and astrocytes in vivo following viral induced inflammation. Furthermore, prolonged MHC expression subsequent to viral clearance implies a potential for ongoing presentation.
Assuntos
Astrócitos/metabolismo , Encefalomielite/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes MHC da Classe II/genética , Genes MHC Classe I/genética , Microglia/metabolismo , Animais , Astrócitos/patologia , Células Cultivadas , Encefalomielite/patologia , Encefalomielite/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Inflamação/patologia , Interferon gama/análise , Interferon gama/biossíntese , Masculino , Vírus Elberfeld do Camundongo , Camundongos , Camundongos Transgênicos , Microglia/patologiaRESUMO
In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.
Assuntos
Glioblastoma/diagnóstico , Imidazóis , Luciferases de Renilla , Substâncias Luminescentes , Pirazinas , Animais , Linhagem Celular Tumoral , Feminino , Glioblastoma/metabolismo , Humanos , Imidazóis/administração & dosagem , Injeções Intraperitoneais , Injeções Intravenosas , Luciferases de Renilla/biossíntese , Substâncias Luminescentes/administração & dosagem , Camundongos , Camundongos Nus , Pirazinas/administração & dosagem , Especificidade por SubstratoRESUMO
INTRODUCTION: Deferasirox effectively controls liver iron concentration; however, little is known regarding its ability to remove stored cardiac iron. Deferiprone seems to have increased cardiac efficacy compared with traditional deferoxamine therapy. Therefore, the relative efficacy of deferasirox and deferiprone were compared in removing cardiac iron from iron-loaded gerbils. METHODS: Twenty-nine 8- to 10-week-old female gerbils underwent 10 weekly iron dextran injections of 200 mg/kg/week. Prechelation iron levels were assessed in 5 animals, and the remainder received deferasirox 100 mg/kg/D po QD (n = 8), deferiprone 375 mg/kg/D po divided TID (n = 8), or sham chelation (n = 8), 5 days/week for 12 weeks. RESULTS: Deferasirox reduced cardiac iron content 20.5%. No changes occurred in cardiac weight, myocyte hypertrophy, fibrosis, or weight-to-dry weight ratio. Deferasirox treatment reduced liver iron content 51%. Deferiprone produced comparable reductions in cardiac iron content (18.6% reduction). Deferiprone-treated hearts had greater mass (16.5% increase) and increased myocyte hypertrophy. Deferiprone decreased liver iron content 24.9% but was associated with an increase in liver weight and water content. CONCLUSION: Deferasirox and deferiprone were equally effective in removing stored cardiac iron in a gerbil animal model, but deferasirox removed more hepatic iron for a given cardiac iron burden.