RESUMO
Cyanidin, a kind of anthocyanin, has been reported to have chemotherapeutic activities in humans. Human amniotic epithelial cells (hAECs) are considered a potential source of pluripotent stem cells. hAECs have been used as a novel tool in regenerative cellular therapy and cell differentiation studies. In this study, to explore the effects of cyanidin-3-O-glucoside (Cy3G) on hAECs and their mechanisms, we investigated the transcriptomic changes in the Cy3G-treated cells using microarray analysis. Among the differentially expressed genes (Fold change > 1.1; p-value < 0.05), 109 genes were upregulated and 232 were downregulated. Ratios of upregulated and downregulated genes were 0.22% and 0.47% of the total expressed genes, respectively. Next, we explored the enriched gene ontology, i.e., the biological process, molecular function, and cellular component of the 37 upregulated (>1.3-fold change) and 124 downregulated (<1.3-fold change) genes. Significantly enriched biological processes by the upregulated genes included "response to muscle activity," and the genes involved in this gene ontology (GO) were Metrnl and SRD5A1, which function in the adipocyte. On the other hand, the cell cycle biological process was significantly enriched by the downregulated genes, including some from the SMC gene family. An adipogenesis-associated gene DDX6 was also included in the cell cycle biological process. Thus, our findings suggest the prospects of Cy3G in modulating adipocyte differentiation in hAECs.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Adipocinas/biossíntese , Antocianinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Adipocinas/genética , Âmnio , Diferenciação Celular/genética , Células Epiteliais/citologia , Humanos , Proteínas de Membrana/genéticaRESUMO
BACKGROUND: Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, PCa recurrence and progression rates during ADT are high. Until now, there has been no evidence regarding when progression begins. This study evaluated the gene expression of intraprostatic androgen receptor (AR) and steroidogenic enzymes in the early stages of ADT. METHODS: Prostate tissue samples were taken from PCa patients with urinary retention who received ADT (ADT-PCa; n = 10) and were further subgrouped into ADT ≤12 months (n = 4) and ADT > 12 months (n = 6). The ADT-PCa tissues were then compared with BPH (n = 12) and primary (no treatment) PCa tissues (n = 16). mRNA for gene expression analysis of AR and steroidogenic enzymes was extracted from formalin-fixed paraffin embedded (FFPE) tissues and analyzed by real-time PCR. Protein expression was evaluated by immunohistochemistry with specific antibodies. RESULTS: AR gene expression was higher in the ADT-PCa group than in the BPH or primary PCa group. Both the ADT ≤12 and > 12 months subgroups had significantly higher relative gene expression levels of AR (p < 0.01 and 0.03, respectively) than the primary PCa group. In the ADT-PCa group, AR protein expression showed an increasing trend in the ADT ≤12 months subgroup and was significantly elevated in the ADT > 12 months subgroup compared with the PCa group (100%; p < 0.01). Half (50%) of the patients in the ADT ≤12 months subgroup were found to have upregulation of AR, and one showed upregulation beginning at 3 months of ADT. A trend toward elevated relative gene expression of SRD5A3 was also apparent in the ADT groups. CONCLUSION: AR and steroidogenic enzymes are upregulated in ADT-PCa patients as early as 3 months, without PSA elevation. Steroidogenic enzymes, particularly SRD5A3, were also upregulated before PSA rose.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Hormônio Liberador de Gonadotropina/agonistas , Orquiectomia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/terapia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/análise , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Idoso , Idoso de 80 Anos ou mais , Membro C3 da Família 1 de alfa-Ceto Redutase/análise , Membro C3 da Família 1 de alfa-Ceto Redutase/biossíntese , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Neoplasias da Próstata/química , Neoplasias da Próstata/genética , Receptores Androgênicos/análise , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. METHODOLOGY: Immature Leydig cells isolated from 35 day-old rats were cultured with 30 µM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 µM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. RESULTS AND CONCLUSIONS: In intact Leydig cells, 30 µM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 µM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 µM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 µM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.
Assuntos
Androgênios/biossíntese , Anestésicos Intravenosos/toxicidade , Etomidato/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Anestésicos Intravenosos/farmacologia , Animais , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Meios de Cultura/farmacologia , Citosol/química , Estradiol Desidrogenases/biossíntese , Estradiol Desidrogenases/genética , Etomidato/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios Esteroides Gonadais/farmacologia , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/ultraestrutura , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Microssomos/química , Mitocôndrias/química , Ratos , Ratos Sprague-Dawley , Testículo/citologia , Testículo/crescimento & desenvolvimentoRESUMO
RATIONALE: The implications of the neurosteroid 3α-hydroxy-5α-pregnan-20-one [allopregnanolone (Allo)] in neuropsychiatric disorders have been highlighted in several recent clinical investigations. For instance, Allo levels are decreased in the cerebrospinal fluid (CSF) of patients with posttraumatic stress disorder (PTSD) and major unipolar depression. Neurosteroidogenic antidepressants [i.e., selective brain steroidogenic stimulants (SBSSs)], including fluoxetine and analogs, correct this decrease in a manner that correlates with improved depressive symptoms. Allo positively and allosterically modulates GABA action at postsynaptic and extrasynaptic GABAA receptors. It is synthesized in both the human and rodent brain cortices by principal glutamatergic pyramidal neurons from progesterone by the sequential action of 5α-reductase type I (5α-RI), which is the rate-limiting step enzyme in Allo biosynthesis, and 3α-hydroxysteroid dehydrogenase (3α-HSD), which converts 5α-dehydroprogesterone into Allo. HYPOTHESIS: We thus hypothesized that decreased CSF levels of Allo in depressed patients could reflect a brain dysfunction of 5α-RI. METHODS: In a pilot study of samples from six patients per group [six depressed patients and six nonpsychiatric subjects (NPS)], we studied the expression of 5α-RI messenger RNA (mRNA) in prefrontal cortex Brodmann's area 9 (BA9) and cerebellum from depressed patients obtained from the Maryland Brain Collection at the Maryland Psychiatric Research Center (Baltimore, MD) that were age-matched with NPS. RESULTS: The levels of 5α-RI mRNA were decreased from 25 ± 5.8 in NPS to 9.1 ± 3.1 fmol/pmol neuronal specific enolase (NSE) (t1,10 = 2.7, P = 0.02) in depressed patients. These differences are absent in the cerebellum of the same patients. The levels of neurosteroids were determined in the prefrontal cortex BA9 of depressed patients obtained from the Stanley Foundation Brain Bank Neuropathology Consortium, Bethesda (MD). The BA9 levels of Allo in male depressed patients failed to reach statistical difference from the levels of NPS (1.63 ± 1.01 pg/mg, n = 8, in NPS and 0.82 ± 0.33 pg/mg, n = 5, in nontreated depressed patients). However, depressed patients who had received antidepressant treatment (three patients SSRI and one TCA) exhibited increased BA9 Allo levels (6.16 ± 2.5 pg/mg, n = 4, t1,9 = 2.4, P = 0.047) when compared with nontreated depressed patients. CONCLUSIONS: Although in a small number of patients, this finding is in-line with previous reports in the field that have observed an increase of Allo levels in CSF and plasma of depressed patients following antidepressant treatment. Hence, the molecular mechanisms underlying major depression may include a GABAergic neurotransmission deficit caused by a brain Allo biosynthesis downregulation, which can be normalized by SBSSs.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Transtorno Depressivo Maior/genética , Córtex Pré-Frontal/enzimologia , Adulto , Transtorno Depressivo Maior/líquido cefalorraquidiano , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Pregnanolona/líquido cefalorraquidiano , Células Piramidais/enzimologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Suicídio , Bancos de Tecidos , Adulto JovemRESUMO
RATIONALE: Underage drinking is a leading public health problem in developed countries. An increasing proportion of adolescents consume alcoholic beverages every weekend. Increased anxiety, irritability, and depression among adolescents may induce them to seek for the anxiolytic and rewarding properties of alcohol. Allopregnanolone (AlloP) shares rewarding effects of ethanol and modulates ethanol intake. The rate-limiting enzyme in the biosynthesis of AlloP is steroid 5α-reductase (5α-R), which is expressed as three isozymes, 5α-R1, 5α-R2, and 5α-R3. OBJECTIVE: The objective of this study was to quantify the expression levels of 5α-R isozymes in prefrontal cortex (PFC) of adolescent male rats after three different regimes of ethanol administration. METHODS: Adolescent male Wistar rats were administered with ethanol (4 g/kg) or saline intraperitoneally for 1 day (acute), for 7 days (chronic), or every 72 h for 14 days (chronic intermittent). Messenger (m)RNA and protein levels of 5α-R isozymes were measured by quantitative RT-PCR and Western blot, respectively. RESULTS: Ethanol significantly increased mRNA and protein levels of 5α-R1, 5α-R2, and 5α-R3 in the three different regimes of ethanol administration, being higher in the chronic intermittent regime in comparison with the others. CONCLUSIONS: The expression of the AlloP-biosynthetic enzyme 5α-Rs increases in the prefrontal cortex of adolescent male rats under acute, chronic, and chronic intermittent regime of ethanol administration. The latter is very interesting because it mimics the teenage drinking behavior.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/biossíntese , Alcoolismo/enzimologia , Alcoolismo/metabolismo , Animais , Consumo Excessivo de Bebidas Alcoólicas/enzimologia , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Isoenzimas/biossíntese , Masculino , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/enzimologia , Ratos , Ratos WistarRESUMO
We examined whether variants in genes related to sex hormone biosynthesis and metabolism were associated with hypospadias in humans. We examined 332 relatively common tag single-nucleotide polymorphisms (tagSNPs) in 20 genes. Analyses included 633 cases (84 mild, 322 moderate, 212 severe and 15 undetermined severity) and 855 population-based non-malformed male controls born in California from 1990 to 2003. We used logistic regression models to estimate odds ratios (OR) and 95% confidence intervals (CI) for each SNP. Several of the 332 studied SNPs had p < 0.01: one in CYP3A4, four in HSD17B3, one in HSD3B1, two in STARD3, 10 in SRD5A2 and seven in STS. In addition, haplotype analyses gave several associations with p < 0.01. For HSD17B3, 14-SNP and 5-SNP blocks had ORs of 1.5 (95% CI 1.1, 2.0, p < 0.001) and 2.8 (95% CI 1.6, 4.8, p < 0.001) respectively. For SRD5A2, 9-SNP, 3-SNP and 8-SNP blocks had ORs of 1.7 (95% CI 1.3, 2.2, p < 0.001), 1.4 (95% CI 1.1, 1.8, p = 0.008) and 1.5 (95% CI 1.2, 1.9, p = 0.002) respectively. Our study indicates that several genes that contribute to sex hormone biosynthesis and metabolism are associated with hypospadias risk.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Hormônios Esteroides Gonadais/genética , Hipospadia/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Predisposição Genética para Doença , Variação Genética , Genótipo , Hormônios Esteroides Gonadais/biossíntese , Hormônios Esteroides Gonadais/metabolismo , Humanos , Hipospadia/epidemiologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Pênis/anormalidades , Pênis/anatomia & histologia , Polimorfismo de Nucleotídeo Único , Progesterona Redutase/biossíntese , Progesterona Redutase/genética , Risco , Esteroide Isomerases/biossíntese , Esteroide Isomerases/genética , Esteril-Sulfatase/biossíntese , Esteril-Sulfatase/genéticaRESUMO
In both epididymis and prostate the dynamic cross-talk between the cells is hormonally regulated and, in part, through direct cell-to-cell interactions. Functionality of the male reproductive organs may be affected by exposure to specific chemicals, so-called 'reprotoxicants'. In this study we tested whether early postnatal and prepubertal exposure to anti-androgen flutamide altered the expression of adherens junction genes encoding E-cadherin (CDH1) and ß-catenin (CTNNB1) in adult pig epididymis and prostate. In addition, the expression of mRNAs and proteins for 5α-reductase (ST5AR2) and aromatase (CYP19A1) were examined to show whether flutamide alters metabolism of testosterone. Thus, flutamide was injected into male piglets between Days 2 and 10 and between Days 90 and 98 postnatally (PD2 and PD90; 50 mg/kg bw), tissues that were obtained on postnatal Day 270. To assess the expression of the genes and proteins, real-time RT-PCR and Western blot were performed respectively. Moreover, adherens junction proteins were localized by immunohistochemistry. In response to flutamide, CDH1 and CTNNB1 expressions were down-regulated along the epididymis, mostly in PD2 group (p < 0.001, p < 0.01). In the prostate, CDH1 mRNA and protein expressions were significantly down-regulated (p < 0.01), whereas CTNNB1 mRNA was slightly up-regulated in both flutamide-treated groups. CTNNB1 protein level was markedly elevated in both PD2 (p < 0.001) and PD90 (p < 0.01) groups. In the epididymis, the expression of ST5AR2 and CYP19A1 was down- and up-regulated, respectively (p < 0.05), whereas in the prostate evident decrease in CYP19A1 expression (p < 0.001, p < 0.01, p < 0.05) was demonstrated. In both tissues, membranous immunolocalization of CTNNB1 suggests its involvement in cell-cell adhesion. Overall, flutamide administration resulted in suppression of androgen action in the epididymis and prostate leading to deregulation of CDH1 and CTNNB1 gene expressions which is probably caused by the alterations in the expression of ST5AR2 and CYP19A1 in both reproductive organs.
Assuntos
Antagonistas de Androgênios/farmacologia , Proteínas Cdh1/biossíntese , Flutamida/farmacologia , Próstata/metabolismo , beta Catenina/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Junções Aderentes/metabolismo , Androgênios/metabolismo , Animais , Aromatase/biossíntese , Aromatase/genética , Proteínas Cdh1/genética , Adesão Celular , Comunicação Celular/efeitos dos fármacos , Epididimo/citologia , Epididimo/metabolismo , Masculino , Próstata/citologia , RNA Mensageiro/biossíntese , Suínos , Testosterona/metabolismo , beta Catenina/genéticaRESUMO
Several studies have demonstrated that the presence of stressors during pregnancy induces adverse effects on the neuroendocrine system of the offspring later in life. In the present work, we investigated the effects of early programming on the male reproductive system, employing a prenatal stress (PS) paradigm. This study found that when pregnant dams were placed in a plastic restrainer three times a day during the last week of pregnancy, the offspring showed reduced anogenital distance and delayed testicular descent. Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels were decreased at postnatal day (PND) 28 and testosterone was decreased at PND 75. Increased testosterone plus dihydrotestosterone (T + DHT) concentrations correlated with increased testicular 5α Reductase-1 (5αR-1) mRNA expression at PND 28. Moreover, PS accelerated spermatogenesis at PND 35 and 60, and increased mean seminiferous tubule diameter in pubertal offspring and reduced Leydig cell number was observed at PND 35 and 60. PS offspring had increased androgen receptor (AR) mRNA level at PND 28, and at PND 35 had increased the numbers of Sertoli cells immunopositive for AR. Overall, the results confirm that stress during gestation can induce long-term effects on the male offspring reproductive system. Of particular interest is the pre-pubertal imbalance of circulating hormones that probably trigger accelerated testicular development, followed by an increase in total androgens and a decrease in testosterone concentration during adulthood. Exposure to an unfavourable intrauterine environment might prepare for harsh external conditions by triggering early puberty, increasing reproductive potential.
Assuntos
Exposição Materna , Efeitos Tardios da Exposição Pré-Natal , Estresse Psicológico , Testículo/crescimento & desenvolvimento , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Animais , Di-Hidrotestosterona/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/sangue , Masculino , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Androgênicos/metabolismo , Restrição Física , Espermatogênese , Testosterona/sangueRESUMO
Endometriosis is defined as the presence of endometrial glands and stroma outside the uterine cavity. This disease is associated with diminished protective effects of progesterone, which are usually explained by inadequate activation of progesterone receptors and also enhanced pre-receptor metabolism of progesterone. Endometriosis is often treated with progestins, which act as progesterone receptor agonists, although their exact mechanisms of action are not completely understood. The aim of the present study was to investigate how the progestins medroxyprogesterone acetate, dydrogesterone and dienogest, as well as progesterone, impact on the expression of genes of pre-receptor progesterone metabolism in Z-12 epithelial cell line, a model system of peritoneal endometriosis. Our data demonstrate that these progestins affect local pre-receptor metabolism to a different extent. The most favorable effects were seen for dydrogesterone and dienogest, where the first, dydrogesterone, significantly reduced SRD5A1, AKR1C2 and AKR1C3 expression, and additionally had a nonsignificant impact on progesterone receptor B (PR-B) protein levels. This might slow down the first step of pre-receptor metabolism, the conversion of progesterone to 5α-dihydroprogestrone by SRD5A1, and it might also affect further reduction of 3-keto and 20-keto groups catalyzed by AKR1C2 and AKR1C3. Similarly favorable effects were seen for dienogest, which promoted significant reduction of AKR1C1 and AKR1C2 expression and also showed no effect on PR-B protein levels. Different effects were seen for progesterone, which significantly decreased SRD5A1, PR-B and HSD17B2 protein levels. In contrast, treatment with medroxyprogesterone acetate resulted in increased AKR1C1 expression and decreased levels of PR-B, which might lead to enhanced progesterone metabolism and reduced signaling through progesterone receptors. Altogether, our data in this Z-12 cell model suggest that the beneficial effects of treatment with progestin observed in endometriosis patients might arise from decreased pre-receptor metabolism of the protective progesterone by the SRD5A1 and AKR1C enzymes.
Assuntos
20-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Endométrio/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Hidroxiprostaglandina Desidrogenases/biossíntese , Proteínas de Membrana/biossíntese , Progestinas/farmacologia , Receptores de Progesterona/biossíntese , 20-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Didrogesterona/farmacologia , Endometriose/genética , Endometriose/metabolismo , Endométrio/metabolismo , Células Epiteliais/metabolismo , Estradiol Desidrogenases/genética , Estradiol Desidrogenases/metabolismo , Feminino , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Hidroxiesteroide Desidrogenases/biossíntese , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroide Desidrogenases/metabolismo , Acetato de Medroxiprogesterona/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nandrolona/análogos & derivados , Nandrolona/farmacologia , Progesterona/farmacologia , RNA Mensageiro/genética , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
Androgen receptor (AR) signaling persists in castration-resistant prostate carcinomas (CRPC), because of several mechanisms that include increased AR expression and intratumoral androgen metabolism. We investigated the mechanisms underlying aberrant expression of transcripts involved in androgen metabolism in CRPC. We compared gene expression profiles and DNA copy number alteration (CNA) data from 29 normal prostate tissue samples, 127 primary prostate carcinomas (PCa), and 19 metastatic PCas. Steroidogenic enzyme transcripts were evaluated by quantitative reverse transcriptase PCR in PCa cell lines and circulating tumor cells (CTC) from CRPC patients. Metastatic PCas expressed higher transcript levels for AR and several steroidogenic enzymes, including SRD5A1, SRD5A3, and AKR1C3, whereas expression of SRD5A2, CYP3A4, CYP3A5, and CYP3A7 was decreased. This aberrant expression was rarely associated with CNAs. Instead, our data suggest distinct patterns of coordinated aberrant enzyme expression. Inhibition of AR activity by itself stimulated AKR1C3 expression. The aberrant expression of the steroidogenic enzyme transcripts was detected in CTCs from CRPC patients. In conclusion, our findings identify substantial interpatient heterogeneity and distinct patterns of dysregulated expression of enzymes involved in intratumoral androgen metabolism in PCa. These steroidogenic enzymes represent targets for complete suppression of systemic and intratumoral androgen levels, an objective that is supported by the clinical efficacy of the CYP17 inhibitor abiraterone. A comprehensive AR axis-targeting approach via simultaneous, frontline enzymatic blockade, and/or transcriptional repression of several steroidogenic enzymes, in combination with GnRH analogs and potent antiandrogens, would represent a powerful future strategy for PCa management.
Assuntos
Androgênios/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/enzimologia , 3-Hidroxiesteroide Desidrogenases/biossíntese , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Androgênios/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Mineração de Dados , Humanos , Hidroxiprostaglandina Desidrogenases/biossíntese , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Imuno-Histoquímica , Masculino , Metástase Neoplásica , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Orquiectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , TranscriptomaRESUMO
Tumor cell proliferation and progression of breast cancer are influenced by female sex steroids. However, not all breast cancer patients respond to aromatase inhibitors (AI), and many patients become unresponsive or relapse. Recent studies demonstrate that not only estrogens but also androgens may serve as regulators of estrogen-responsive as well as estrogen-unresponsive human breast cancers. However, the mechanism underlying these androgenic actions has remained relatively unknown. Therefore, in this study, we evaluated the effects of AI upon the expression of enzymes involved in intratumoral androgen production including 17ß-hydroxysteroid dehydrogenase type 5 (17ßHSD5), 5α-reductase types 1 and 2 (5αRed1 and 5αRed2) as well as androgen receptor (AR) levels and correlated the findings with therapeutic responses including Ki67 labeling index (Ki67). Eighty-two postmenopausal invasive ductal carcinoma patients were enrolled in CAAN study from November 2001 to April 2004. Pre- and post-treatment specimens of 29 cases were available for this study. The status of 17ßHSD5, 5αRed1, 5αRed2, and Ki67 in pre- and post-treatment specimens were evaluated. The significant increments of 5αRed2 as well as AR were detected in biological response group whose Ki67 LI decreased by more than 40% of the pre-treatment level. This is the first study demonstrating an increment of 5αRed2 and AR in the group of the patients associated with Ki67 decrement following AI treatment. These results suggest that increased 5αRed2 and AR following AI treatment may partly contribute to reduce the tumor cell proliferation through increasing intratumoral androgen concentrations and its receptor.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/biossíntese , Androstadienos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal de Mama/tratamento farmacológico , Celecoxib , Feminino , Humanos , Imuno-Histoquímica , Letrozol , Terapia Neoadjuvante , Nitrilas/administração & dosagem , Pirazóis/administração & dosagem , Receptores Androgênicos/biossíntese , Sulfonamidas/administração & dosagem , Resultado do Tratamento , Triazóis/administração & dosagemRESUMO
We previously identified BASP1 and SRD5A2 as novel hepatocellular carcinoma (HCC) methylation markers from among more than 10,000 screened genes. The present study aimed to improve the diagnostic potential of these genes. We compared the methylation status at distinct regions of the BASP1 and SRD5A2 genes using quantitative methylation-specific PCR, in 46 sets of HCC and corresponding non-tumor liver tissues. We also examined how their epigenetic status affected transcript levels in tissues and several hepatoma cell lines. We found that BASP1 and SRD5A2 loci were methylated in greater than 50% of the HCC tissues. Inverse correlations were identified between the methylation status and transcript levels in the tissues. Assessment of CpG island methylation rate of BASP1 and SRD5A2 resulted in different diagnostic powers for discriminating HCC even in the same CpG island. A combination analysis of BASP1 and SRD5A2 resulted in the optimum diagnostic performance (84.8% sensitivity and 91.3% specificity) with a maximal area under the receiver operating characteristic curve of 0.878. Even in patients with early HCC (well-differentiated, TNM stage I and small in diameter) and those negative for serum alpha-fetoprotein, combination analysis enabled an accurate diagnosis of HCC. In vitro analysis also showed that BASP1 and SRD5A2 transcripts were epigenetically regulated by methylation and acetylation. These results suggest that combined analysis of methylated BASP1 and SRD5A2 may prove useful in the accurate diagnosis of HCC, especially early HCC.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Metilação de DNA , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Idoso , Ilhas de CpG , Primers do DNA/genética , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prostate cancer often relapses during androgen-depletion therapy, even under conditions in which a drastic reduction of circulating androgens is observed. There is some evidence that androgens remain present in the tissues of hormone-refractory prostate cancers (HRPC), and enzymes involved in the androgen and steroid metabolic pathway are likely to be active in HRPC cells. We previously carried out a genome-wide gene expression profile analysis of clinical HRPC cells by means of cDNA microarrays in combination with microdissection of cancer cells and found dozens of transactivated genes. Among them, we here report the identification of a novel gene, SRD5A2L, encoding a putative 5 alpha-steroid reductase that produces the most potent androgen, 5 alpha-dihydrotestosterone (DHT), from testosterone. Liquid chromatography-tandem mass spectrometry analysis following an in vitro 5 alpha-steroid reductase reaction validated its ability to produce DHT from testosterone, similar to type 1 5 alpha-steroid reductase. Because two types of 5 alpha-steroid reductase were previously reported, we termed this novel 5 alpha-steroid reductase 'type 3 5 alpha-steroid reductase' (SRD5A3). Reverse transcription-polymerase chain reaction and northern blot analyses confirmed its overexpression in HRPC cells, and indicated no or little expression in normal adult organs. Knockdown of SRD5A3 expression by small interfering RNA in prostate cancer cells resulted in a significant decrease in DHT production and a drastic reduction in cell viability. These findings indicate that a novel type 3 5 alpha-steroid reductase, SRD5A3, is associated with DHT production and maintenance of androgen-androgen receptor-pathway activation in HRPC cells, and that this enzymatic activity should be a promising molecular target for prostate cancer therapy.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias da Próstata/enzimologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Animais , Células COS , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Di-Hidrotestosterona/metabolismo , Humanos , Masculino , Neoplasias da Próstata/genética , RNA Interferente Pequeno/genética , Testosterona/metabolismo , TransfecçãoRESUMO
In diabetes, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis causes effects such as elevation of corticotropin (ACTH) and glucocorticoids. Cholecystokinin and its receptors are involved in the HPA axis and influence the regulation of the HPA axis. We examined adrenocortical function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus, that lack the cholecystokinin A receptor. We measured adrenal weight, plasma ACTH, serum and urinary corticosterone, and serum leptin in OLETF rats at 5 to 36 weeks of age. Messenger RNA (mRNA) expression of 11beta-hydroxysteroid dehydrogenase and 5alpha-reductase type 1 in adrenal glands of the rats were examined. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. In OLETF rats at 32 to 36 weeks of age, plasma ACTH was significantly higher (P < .001); serum corticosterone and 24-hour urinary corticosterone were significantly lower (P < .005); and adrenal weight was significantly lower (P < .005) than those in LETO rats. At the same ages, serum leptin in OLETF rats was significantly higher (P < .001) than that in LETO rats. In the younger OLETF rats, these changes were not observed. Overall, there was an inverse correlation between serum corticosterone and serum leptin (r = -0.374, P < .0005), whereas there was a positive correlation between plasma ACTH and serum leptin (r = 0.654, P < .0001). At 5 and 36 weeks of age, mRNA expression of 5alpha-reductase type 1 in the adrenal gland of OLETF rats was significantly higher (P < .05) than that of LETO rats, whereas there was no significant difference in mRNA expressions of 11beta-hydroxysteroid dehydrogenase types 1 and 2. We showed that adrenocortical insufficiency and adrenal atrophy were acquired in OLETF rats, and the possibility of elevated serum leptin relates to this phenomenon.
Assuntos
Insuficiência Adrenal/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Córtex Suprarrenal/patologia , Testes de Função do Córtex Suprarrenal , Glândulas Suprarrenais/metabolismo , Insuficiência Adrenal/metabolismo , Insuficiência Adrenal/patologia , Hormônio Adrenocorticotrópico/sangue , Envelhecimento/fisiologia , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Corticosterona/sangue , Corticosterona/urina , Primers do DNA , Diabetes Mellitus Tipo 2/metabolismo , Insulina/sangue , Leptina/sangue , Tamanho do Órgão/fisiologia , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos OLETF , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In many androgen target tissues, testosterone is reduced to the more potent androgen, dihydrotestosterone, by steroid 5alpha-reductase. Two isoforms of 5alpha-reductase, type 1 and type 2, have been cloned. They are differentially expressed and regulated. To determine the mechanisms of regulation of 5alpha-reductase type 1 expression, we have cloned its 5'upstream region and defined its promoter. The proximal 5'upstream region of 5alpha-reductase type 1 displays all the features of a CpG island and has numerous Sp1 binding sites. By transient transfection assays, we have identified a bidirectional promoter activity in this region; this activity was highest in the negative orientation, in the direction of the methyltransferase Nsun2 (predicted) gene. Promoter activity, in either orientation, was lost in Sp1 deficient cells but was rescued following co-transfection with a Sp1 expression vector. Thus, the 5'upstream region of rat 5alpha-reductase type 1 contains a bidirectional promoter with an activity that is Sp1-dependent.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Ilhas de CpG/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Elementos de Resposta/fisiologia , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/fisiologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Androgênios/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Biblioteca Genômica , Metiltransferases/biossíntese , Metiltransferases/genética , Ratos , Fator de Transcrição Sp1/genética , Testosterona/metabolismoRESUMO
17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 5alpha-reductase isoenzymes play a crucial role in the formation and metabolism of sex steroids. Not only the key androgens testosterone and dihydrotestosterone but also their precursors are potent activators of the androgen receptor and are, therefore, likely to act as determinants of male sexual differentiation and maturation in a differentially regulated way. The aim of the present study was to relatively quantify the expression of the mRNA of 17beta-HSD isoenzymes, namely, type 1, 2, 3, 4, 5, 7, and 10, together with the 5alpha-reductase type 1 and 2, and the androgen receptor in normal human males and females. RNA was isolated from peripheral blood cells of both sexes and from genital skin fibroblasts (GSFs) of two different localizations (foreskin and scrotal skin) obtained from phenotypically normal males. mRNA expression was semi-quantified by quantitative reverse-transcriptase polymerase chain reaction with the LightCycler Instrument (Roche). The examined enzymes show statistically significant differences in their transcription pattern between the blood and the GSF RNA samples. Within the GSF samples, there are also significant variations between the two examined localizations in the transcription of 17beta-HSD type 1, 2, 4, and 5 as well as for the androgen receptor. We found large interindividual variation of enzyme transcription patterns in all investigated tissues. In peripheral blood cells, no sex-specific differences were seen. We conclude that sex steroid enzymes are expressed not only in genital primary target tissues but also in peripheral blood. The expression in different target tissues may contribute to both the individual sexual and tissue-specific phenotype in humans.
Assuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Hormônios Esteroides Gonadais/biossíntese , Receptores Androgênicos/biossíntese , 17-Hidroxiesteroide Desidrogenases/sangue , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/sangue , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Prepúcio do Pênis/metabolismo , Humanos , Lactente , Isoenzimas/biossíntese , Isoenzimas/sangue , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Receptores Androgênicos/sangue , Fatores SexuaisRESUMO
BACKGROUND: High-fat diet is a major risk factor for prostate cancer. 5alpha-reductases are potential targets of dietary fat. METHODS: Male ACI/Seg rats given either a low-fat or a high-fat diet at weaning or adulthood were sacrificed at 2, 4, and 10 weeks after dietary treatment. Prostate 5alpha-reductase mRNAs, plasma androgens, food consumption, prostate, and body weight were determined. RESULTS: Prostate 5alpha-reductase-2 mRNA and plasma dihydrotestosterone levels were elevated at 2 weeks, and prostate weight was increased at 10 weeks in neonatal rats fed the high-fat diet. Animals fed the high-fat diet consumed more calories in the first 4 weeks. 5alpha-reductase-1 mRNA, plasma testosterone, and body weight were not different between the two dietary groups. These dietary effects were not observed in adult rats fed the same diets. CONCLUSION: A high-dietary fat and caloric intake upregulates prostate 5alpha-reductase-2 gene expression, and stimulates prostate growth in neonatal, but not adult rats.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Gorduras na Dieta , Ingestão de Energia , Próstata/crescimento & desenvolvimento , Neoplasias da Próstata/etiologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Peso Corporal , Masculino , Ratos , Fatores de Risco , Testosterona/sangue , Regulação para CimaRESUMO
We studied the expression of type 1 (5alpha-R1) and type 2 (5alpha-R2) 5alpha-reductase isozymes (5alpha-R) and their regulation by dihydrotestosterone (DHT) in the prefrontal cortex of male and female rats during postnatal sexual differentiation of the central nervous system (CNS), using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis. We found a higher expression of 5alpha-R2, which is considered a masculinizing enzyme, in the female versus male CNS, and observed sexual dimorphism in the regulation of both 5alpha-R isozymes by DHT. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , Animais Recém-Nascidos/metabolismo , Química Encefálica/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , RNA Mensageiro/biossíntese , Animais , Eletroforese Capilar , Feminino , Isoenzimas/biossíntese , Masculino , Oligonucleotídeos/farmacologia , Córtex Pré-Frontal/enzimologia , Ratos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres SexuaisRESUMO
OBJECTIVE: 5alpha-reductase enzymes reduce testosterone (T) to the most potent androgen dihydrotestosterone (DHT). Two isoenzymes are known to day. While the type 2-enzyme (5RII) is predominantly expressed in male genital tissues and mutations are known to cause a severe virilization disorder in genetic males, the role of the type 1-enzyme (5RI) in normal male androgen physiology is unclear. We investigated whether 5RI is transcribed in normal male genital skin fibroblasts (GSFs) and if the transcription is regulated by age or by androgens themselves. METHODS: GSF from 14 normally virilized males of different ages, ranging from 8 months to 72 years, obtained at circumcision were cultured. Total RNA was isolated after incubation for 48 h with 100 nM T or without androgens. Each sample was amplified in triplicate by real-time PCR with porphobilinogen desaminase as a housekeeping gene used for semiquantification. Selected cultures were analyzed after incubation with 10 and 100 nM T and 1 and 100 nM DHT for 24, 48 and 120 h. RESULTS: 5RI was transcribed in all investigated samples with a 4.5-fold variability in the mRNA concentration of different individuals. However, neither age-related regulation nor significant influence of T or DHT on the transcription rate was discovered. CONCLUSION: Since 5RI is abundantly transcribed in GSFs, we hypothesize that this isoenzyme may play important roles in the androgen physiology of normally virilized males and may contribute to masculinization in 5RII-deficient males at the time of puberty.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/deficiência , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/fisiologia , Genitália Masculina/enzimologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/biossíntese , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Adulto , Fatores Etários , Idoso , Criança , Pré-Escolar , Fibroblastos , Humanos , Lactente , Isoenzimas , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Diferenciação Sexual/fisiologia , Testosterona/metabolismo , Transcrição GênicaRESUMO
Androgen transformation into estrogens through the aromatase enzyme, occurring in the rat hypothalamus during fetal life, leads to male-specific sexual differentiation of brain. Aromatase shows a peak of expression and activity in a limited period during late gestation; however, the possible dimorphism in its expression during embryogenesis is unclear. One of the mechanisms controlling tissue-specific aromatase expression might be the formation of transcript variants, that differ in the 5'-untranslated regions (5'-UTR). Exon If is the major 5'-UTR used in rodent hypothalamic-preoptic area, with low amounts of other variants encoded by different exons I also present. Another enzymatic conversion, possibly involved in brain differentiation, is the 5 alpha-reduction of Testosterone to DHT, catalyzed by two 5 alpha-reductases (5 alpha-R type1 and 2). Aim of the present study is to evaluate, in parallel, by semiquantitative RT-PCR, the dimorphic profile of the three enzymes and the pattern of the brain-specific aromatase expression in male and female rats from gestation-day 16 to postnatal-day 5 (or 15 only for 5 alpha-R1). It has been observed that, in both sexes, 5 alpha-R1 is significantly higher around birth than prenatally, and that 5 alpha-R2 expression appears to be higher in males than in females, particularly just after birth. Moreover, aromatase has two expression peaks, that are male-specific, before and after birth; only exon If is used in males, while different transcripts might be present in females postnatally. It is concluded that rodent brain sexual differentiation probably involves the activation of both 5 alpha-R2 and aromatase enzymes in a sex- and time-specific pattern.