RESUMO
BACKGROUND: GnRH analogs are widely used as neoadjuvant agents for radiotherapy in prostate cancer (PCa) patients, with well-documented effects in reducing tumor bulk and increasing progression-free survival. GnRH analogs act locally in the prostate by triggering apoptosis of PCa cells via activation of the GnRH receptor (GnRHR). During PCa progression, the distribution of GnRHR within the cell is altered, with reduced expression in the cell membrane and remaining sequestered in the endoplasmic reticulum. Pharmacoperone IN3 is able to relocalize GnRHR to the cell membrane. The aim of this study was to evaluate the effect of radiation on PCa cells pretreated with leuprolide, alone or in combination with IN3, as radiosensitizers. MATERIAL AND METHODS: PC3 and human PCa primary cell cultures were treated with IN3 for 24 h, followed by different doses of leuprolide for 48 h and, finally, single doses of radiation (3, 6, and 9 Gy). After radiation, cell survival, apoptosis, cell cycle distribution, and colony growth were evaluated. RESULTS: Radiation reduced cell survival and increased apoptosis in a dose-dependent manner. This effect was also directly related to leuprolide concentration. Pretreatment with IN3 enhanced apoptosis and decreased cell survival, also observing a higher proportion of cells arrested in G2. CONCLUSION: Neoadjuvant leuprolide increases radiation-mediated apoptosis of PCa cells. This effect was enhanced by pretreatment with pharmacoperone IN3. Clinical use of IN3 as a radiosensitizer combined with androgen deprivation therapy to improve survival of patients with PCa remains to be evaluated.
Assuntos
Neoplasias da Próstata , Antagonistas de Androgênios , Hormônio Liberador de Gonadotropina , Humanos , Leuprolida/farmacologia , Masculino , Próstata , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/radioterapia , Radiossensibilizantes/farmacologia , Receptores LHRHRESUMO
Cancer stem cells (CSCs) have the ability to self-renew and differentiate to give rise to heterogeneous phenotype of the tumor cells. It is believed that these cells are involved in metastasis, recurrence and therapy resistance in various cancers. CSCs have been identified in prostate cancer (PCa), one of the most diagnosed malignancies in men over the world, for which chemotherapy resistance is a major problem in the treatment of castration-resistant advanced stages. Molecular signatures, gene expression and functional features have been reported for PCa CSCs. Most data come from cell lines which may not represent the actual tumor. In the present work, a CSCs enriched population obtained from PCa explants was functionally characterized and analyzed for drug resistance. Tumorsphere cultures positive for ABCG2 transporter, CD133, CD44, cytokeratins 5 and 18 (CK5 and CK18) and negatives for androgen receptor (AR) and prostate-specific antigen (PSA) showed higher clonogenic capacity, holoclone-forming ability, colony-forming capacity in soft agar and lower proliferative and apoptotic rate than control adherent cell cultures. Furthermore, exposing tumorsphere cultures to ABCG2 substrate drugs resulted in a high survival rate compared with control PCa cells. This high drug resistance was decreased using a selective inhibitor of ABCG2. According to these results, tumorspheres from PCa explants showed a functional stem phenotype and a marked drug resistance, probably mediated by high expression of the ABCG2 transporter, which might be considered as a suitable therapeutic target for CSCs.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Esferoides Celulares/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Topotecan/farmacologia , Células Tumorais CultivadasRESUMO
Gonadotropin-releasing hormone (GnRH) agonists are widely used for the treatment of advanced prostate cancer (PCa). Agonists activate the GnRH receptor (GnRH-R), triggering apoptosis in PCa cells. In gonadotropes, the amount of GnRH-R in the plasma membrane is regulated by protein folding and endoplasmic reticulum retention, mechanisms that can be overcome by the pharmacoperone IN3. Our aim was to describe the intracellular distribution of GnRH-R in PCa cells and its relation to response to GnRH analog treatments. The expressions of GnRH-R in PCa biopsies were evaluated by immunohistochemistry and the intracellular distribution was determined by immunofluorescence in primary cell cultures from human PCa samples. Cultured cells were pretreated with IN3 and then with leuprolide. Cell survival was evaluated by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) thiazolyl blue formazan and cell cycle and apoptosis by flow cytometry. We observed that the expression of GnRH-R decreased according to malignant progression. Most GnRH-R are located inside the cell, colocalizing with endoplasmic reticulum markers. The treatment with IN3 decreased cellular GnRH-R retention, increasing plasma membrane expression in approximately 60%. Pretreatment with IN3 decreased PCa cell survival compared with leuprolide-alone treatment, primarily because of an increase in apoptosis. We conclude that the response of PCa cells to leuprolide is related to the amount of GnRH-R in the plasma membrane. Therefore, pretreatment evaluation of the amount of these receptors may be a predictor of the outcome of leuprolide treatment in PCa patients. Assessment of systemic IN3 effect would be necessary to determine its utility as an adjuvant treatment in hormone-resistant tumors.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Indóis/farmacologia , Leuprolida/farmacologia , Neoplasias da Próstata/patologia , Piridinas/farmacologia , Receptores LHRH/agonistas , Western Blotting , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/metabolismo , Dobramento de Proteína , Receptores LHRH/biossíntese , Células Tumorais CultivadasRESUMO
Prostate cancer (PCa) is the most frequently diagnosed malignancy in men worldwide. Chemotherapy response is very poor and resistance to hormone-based treatments is frequent in advances stages. Recently, tumor-initiating cells or cancer stem cells (CSCs) have been identified in several cancers, including PCa. These cells are thought to be responsible for therapy resistance, relapse and metastasis. In the present work, enriched populations of CSCs were obtained using a mixed procedure that included differential clone-forming ability, sphere growing induction (prostatospheres) and magnetic-associated cell sorting (MACS). Also, stem marker expression was determined in PCa biopsies of different histological grades and metastasis samples. The signature for stem markers of the isolated CSCs was CD133+/CD44+/ABCG2+/ CD24-. Expression of stem markers (CD133, CD44, and ABCG2) was higher in medium Gleason biopsies than in lower and higher grades, and lymph-node and bone metastasis samples. These results suggest that the CSCs in PCa reach an important number in medium Gleason grades, when the tumor is still confined into the gland. At this stage, the surgical treatment is usually with curative intention. However, an important percentage of patients relapse after treatment. Number and signature of CSCs may be a prognosis factor for PCa recurrence.
Assuntos
Antígenos CD/análise , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/genética , Biomarcadores Tumorais/análise , Biópsia , Neoplasias Ósseas/secundário , Separação Celular , Humanos , Imuno-Histoquímica , Metástase Linfática/patologia , Masculino , Gradação de Tumores , Metástase Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Neoplasias da Próstata/patologia , Ensaio Tumoral de Célula-TroncoRESUMO
Prostate cancer (PCa) is the most frequently diagnosed malignancy in men worldwide. Chemotherapy response is very poor and resistance to hormone-based treatments is frequent in advances stages. Recently, tumor-initiating cells or cancer stem cells (CSCs) have been identified in several cancers, including PCa. These cells are thought to be responsible for therapy resistance, relapse and metastasis. In the present work, enriched populations of CSCs were obtained using a mixed procedure that included differential clone-forming ability, sphere growing induction (prostatospheres) and magnetic-associated cell sorting (MACS). Also, stem marker expression was determined in PCa biopsies of different histological grades and metastasis samples. The signature for stem markers of the isolated CSCs was CD133+/CD44+/ABCG2+/ CD24-. Expression of stem markers (CD133, CD44, and ABCG2) was higher in medium Gleason biopsies than in lower and higher grades, and lymph-node and bone metastasis samples. These results suggest that the CSCs in PCa reach an important number in medium Gleason grades, when the tumor is still confined into the gland. At this stage, the surgical treatment is usually with curative intention. However, an important percentage of patients relapse after treatment. Number and signature of CSCs may be a prognosis factor for PCa recurrence.
Assuntos
Humanos , Masculino , Antígenos CD/análise , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/genética , Biópsia , Neoplasias Ósseas/secundário , Separação Celular , Imuno-Histoquímica , Metástase Linfática/patologia , Gradação de Tumores , Metástase Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Neoplasias da Próstata/patologia , Ensaio Tumoral de Célula-Tronco , Biomarcadores Tumorais/análiseRESUMO
BACKGROUND: In several cancer types, expression of multidrug resistance (MDR) proteins has been associated with lack of chemotherapy response. In advanced prostate cancer (PCa) the use of chemotherapy is mainly palliative due to its high resistance. Previously, we described that MDR phenotype in PCa could be related with high basal and drug-induced expression of MDR proteins P-Glycoprotein (P-Gp), MRP1, and LRP. METHODS: Using primary cell cultures from PCa patients, we evaluated the effect of function and expression inhibition of P-Gp, MRP1, and LRP, on cell survival after chemotherapy exposure. Cells were treated with specific MDR protein substrates (docetaxel and mitoxantrone for P-Gp, methotrexate for MRP1 and cisplatin for LRP) and pharmacological inhibitors (cyclosporine A, genistein and 3-aminobenzamide), and cell survival was evaluated trough 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell cycle analysis. MRP1 activity was evaluated by FACS using the specific inhibitor MK571. Cells were transfected with MDR proteins siRNAs and treated with the corresponding substrates. RESULTS: PCa cell resistance to MDR protein substrates was partially reversed, decreasing cell survival in around 20%, by treating primary cell cultures with specific pharmacological inhibitors. PCa cells transfected with siRNAs against MDR proteins decreased cell survival when treated with the corresponding drugs. Docetaxel was the most effective chemotherapeutic drug to induce cell death and decrease survival. CONCLUSION: Low chemotherapy response in PCa could be explained, in part, by over-expression of functional MDR proteins. Expression and function of these proteins should be evaluated to enhance efficacy of docetaxel-based therapies of patients with hormone-resistant PCa.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias da Próstata/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Terapia Combinada , Humanos , Masculino , Fenótipo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
The epithelial-mesenchymal transition (EMT) is considered a key step in tumor progression, where the invasive cancer cells change from epithelial to mesenchymal phenotype. During this process, a decrease or loss in adhesion molecules expression and an increase in migration molecules expression are observed. The aim of this work was to determine the expression and cellular distribution of syndecan-1 and -2 (migration molecules) and E-cadherin and beta-catenin (adhesion molecules) in different stages of prostate cancer progression. A quantitative immunohistochemical study of these molecules was carried out in tissue samples from benign prostatic hyperplasia and prostate carcinoma, with low and high Gleason score, obtained from biopsies archives of the Clinic Hospital of the University of Chile and Dipreca Hospital. Polyclonal specific antibodies and amplification system of estreptavidin-biotin peroxidase and diaminobenzidine were used. Syndecan-1 was uniformly expressed in basolateral membranes of normal epithelium, changing to a granular cytoplasmatic expression pattern in carcinomas. Syndecan-2 was observed mainly in a cytoplasmatic granular pattern, with high immunostaining intensity in areas of low Gleason score. E-cadherin was detected in basolateral membrane of normal epithelia showing decreased expression in high Gleason score samples. beta-Catenin was found in cell membranes of normal epithelia changing its distribution toward the nucleus and cytoplasm in carcinoma samples. We concluded that changes in expression and cell distribution of E-cadherin and beta-catenin correlated with the progression degree of prostate adenocarcinoma, suggesting a role of these molecules as markers of progression and prognosis. Furthermore, changes in the pattern expression of syndecan-1 and -2 indicate that both molecules may be involved in the EMT and tumor progression of prostate cancer.
Assuntos
Caderinas/análise , Transição Epitelial-Mesenquimal , Neoplasias da Próstata/patologia , Sindecana-1/análise , Sindecana-2/análise , beta Catenina/análise , Biomarcadores , Humanos , Imuno-Histoquímica , Masculino , Sindecana-1/fisiologia , Sindecana-2/fisiologiaRESUMO
BACKGROUND: Multidrug resistance (MDR) proteins have been associated with the lack of chemotherapy response. Expression of these proteins has been described in the prostate, but there is no information about their role in the chemotherapy response of prostate cancer (PC). We studied the gene and protein expression of MDR proteins in primary cell cultures from PC tumors and PC cell lines, their relationship with chemotherapy and their effects on cell survival. METHODS: Primary cell cultures from PC were obtained from samples provided by our Institutional Hospital. Cell lines LNCaP, PC3, and DU145 were also examined. Cells were treated during 72 hr with several chemotherapeutic drugs. Protein and mRNA expressions of P-glycoprotein (P-Gp), MRP1 and LRP, before and after drug treatment, were evaluated by RT-PCR and Western blot analyses. The effect on cell survival was evaluated by proliferation assays (MTT), and cell cycle and apoptosis by flow cytometry. RESULTS: Primary PC cultures exhibited higher MDR protein expression and lower drug sensitivity than cell lines, in which P-Gp was not detected. Docetaxel and mitoxantrone displayed the highest apoptotic effect. Exposure to chemotherapeutic drugs increased apoptosis, cell cycle arrest, and MDR expression. Long-term treatment with doxorubicin diminished apoptosis elicited by all drugs examined in this study, suggesting a cross-resistance phenomenon. CONCLUSIONS: Low chemotherapy response observed in PC primary cultures could be explained, in part, by the high levels of MDR proteins (intrinsic MDR phenotype), and also, by their over-expression induced after long-term exposure to drugs (acquired MDR phenotype), which increase treatment resistance. Prostate 69: 1448-1459, 2009. (c) 2009 Wiley-Liss, Inc.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/fisiopatologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Docetaxel , Humanos , Masculino , Metotrexato/farmacologia , Mitoxantrona/farmacologia , Neoplasias da Próstata/patologia , Taxoides/farmacologia , Células Tumorais Cultivadas , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
BACKGROUND: Gonadotropin-releasing-hormone (GnRH) analogs are widely used to block hypothalamic-pituitary-gonadal axis and inhibit blood androgen levels in patients with prostate cancer (PCa). In addition, GnRH analogs induce proliferation arrest and apoptosis through GnRH receptors expressed on the membrane of PCa cells. Possible molecular mechanisms involved in GnRH-mediated apoptosis on prostate cancer cells were studied. METHODS: Primary cultures from PCa and benign prostatic hyperplasia (BPH) (non-malignant control) were derived from samples provided by our Institutional Hospital. Cell cultures were incubated for 24 hr with 20 ng/ml of GnRH agonist Leuprolide (Lp) or antagonist Cetrorelix (Cx). Apoptosis was evaluated by studying the expression of Bax and Bcl-2 and the activation of caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and caspase-3. Also, mRNA level, protein expression and phosphorylation of p53 were studied. RESULTS: Cleaved caspase-8 and -3, but not -9, increased in presence of Lp and Cx in PCa cell cultures. Bax and Bcl-2 mRNA levels showed no changes after GnRH-analog treatments. Only Bax protein showed an increase after Cx treatment in PCa cell cultures. p53 mRNA level was higher in PCa than in BPH cell cultures. Lp and Cx increased p53 expression and phosphorylation in PCa cell cultures. CONCLUSIONS: Apoptosis induced by GnRH analogs seems to be mediated by extrinsic pathway involving p53 phosphorylation. Phosphorylated-p53 might be associated with the increase in apoptotic NGF receptor, p75, previously reported by our laboratory. These findings reinforce the concept of clinical use of GnRH analogs for PCa suggesting that intraprostatic treatment may be more effective.
Assuntos
Adenocarcinoma/tratamento farmacológico , Apoptose/fisiologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/uso terapêutico , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Transurethral needle ablation (TUNA) is an accepted and effective therapy for the treatment of lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH). Prostiva (Medtronic, Shoreview, MN) is the newest-generation device, which includes a new needle design and radio frequency (RF) generator. This device creates temperatures of 120 degrees C and necrotic lesions in less than 2.5 min. Using previously described techniques, we analyzed dynamic, gadolinium-enhanced MRIs to characterize the ablative properties of the new Prostiva RF device. Ten men with LUTS due to BPH were treated with the standard Prostiva manufacturer-recommended protocol. The bladder neck and lateral lobes received treatment based on prostate volume and prostatic urethral length. Gadolinium-enhanced MRI sequences were obtained prior to and 1 week post-treatment. Analyze software (Mayo Clinic Biomedical Imaging Resource, Rochester, MN) was used to evaluate MRIs. New gadolinium defects were seen in all patients following Prostiva treatments. All lesions coalesced within the prostate. No defects were seen beyond the prostate, and the urethra was spared in all patients. The mean volume of necrosis was 7.56 cc, representing a mean of 11.28% of total prostate volume. Dynamic, gadolinium-enhanced MRIs demonstrate new vascular defects representing necrosis caused by Prostiva RF therapy of the prostate. The standard Prostiva RF protocol produces lesions that coalesce to create larger lesions in the bladder neck and lateral lobes. Compared to the TUNA Precision Plus device, the ablative lesions appear comparable while produced with a shorter burn time.
Assuntos
Ablação por Cateter , Gadolínio , Hiperplasia Prostática/patologia , Hiperplasia Prostática/cirurgia , Humanos , Imageamento Tridimensional , Imageamento por Ressonância Magnética , MasculinoRESUMO
7-alpha-Methyl-19-Nortestosterone (MENT) is a synthetic androgen more potent than testosterone (T) and cannot be reduced at 5-alpha position. No important effects of MENT on prostate growth have been reported. However, little is known about the effect of MENT on benign prostatic hyperplasia (BPH) or prostate carcinoma (CaP). We evaluate the effect of MENT, T and dihydrotestosterone (DHT) on secretion, proliferation and gene expression of primary cell cultures from human BPH and CaP. Moreover, the effect of these androgens was examined in the presence of finasteride to determine the influence of the 5-alpha reductase (5-AR) activity on the androgenic potency. BPH and CaP primary cultures were treated with 0, 1, 10 and 100 nM of T, MENT or DHT during 24 and 48 h. Prostate-specific antigen (PSA) was measured by micro particles immunoassay and proliferation rate by spectrophotometric assay (MTT) and by the immunochemical detection of the proliferation marker Ki-67. Gene expression of FGF8b (androgen sensitive gene) was evaluated by semi-quantitative RT-PCR. Results showed that MENT treatments increased PSA secretion and proliferation rate with a potency ranged between T and DHT. Similar effects of MENT were observed in both BPH and CaP cultures. The studies with finasteride showed that in BPH and CaP cells, the conversion of T into DHT significantly contributes to its effect on the proliferation and PSA secretion, and corroborated the resistance of MENT to the 5-AR. The effect of MENT on the gene expression of FGF8b in CaP cells was similar to T and lower than DHT. It is concluded that MENT increases proliferative and secretory activities and gene expression on pathological prostate cells although in less extent than the active metabolite DHT. Furthermore, the fall of endogenous concentration of T during MENT treatment anticipates that this androgen will be of low impact for the prostate.
Assuntos
Nandrolona/análogos & derivados , Próstata/metabolismo , Hiperplasia Prostática/tratamento farmacológico , Hiperplasia Prostática/metabolismo , Androgênios/metabolismo , Androgênios/uso terapêutico , Di-Hidrotestosterona/metabolismo , Di-Hidrotestosterona/farmacologia , Di-Hidrotestosterona/uso terapêutico , Finasterida/metabolismo , Finasterida/farmacologia , Finasterida/uso terapêutico , Humanos , Masculino , Nandrolona/metabolismo , Nandrolona/uso terapêutico , Próstata/patologia , Antígeno Prostático Específico/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testosterona/metabolismo , Testosterona/farmacologia , Testosterona/uso terapêutico , Congêneres da Testosterona/metabolismo , Congêneres da Testosterona/uso terapêuticoRESUMO
Contradictory data have been reported regarding the effect of GnRH agonists and antagonists on cell growth and survival, using prostate cancer-derived cell lines expressing either endogenous or exogenous GnRH receptors. We addressed the issue studying the effect of leuprolide (agonist) and cetrorelix (antagonist) on cell growth, apoptosis and GnRH receptor expression using a primary cell coculture system. Also, binding characteristics of prostate GnRH receptor in this culture system are described. Epithelial and stromal cells were obtained from prostate adenocarcinoma samples and cocultured in a bicameral system. Expression of GnRH receptors was evaluated by semiquantitative RT-PCR (transcript level) and Western blot (protein level). Cell growth was estimated by MTT method and apoptosis by DNA fragmentation using COMET assay. Saturation and competition binding studies were carried out using 125I-GnRH as radioligand. GnRH receptors from cell cultures of prostate cancer exhibited a single class of binding sites with a Kd of 1.11 +/- 0.28 nM and a Bmax of 2.81 +/- 0.37 pmol/mg of membrane protein for GnRH. Leuprolide and cetrorelix showed no effect on GnRH receptor expression. Both analogues showed a significant reduction in cell growth rate and an increase in DNA-fragmented cell number. These effects were dependent on the analogue concentrations (from 5-20 ng/mL). Considering that the culture system used in this work represents more closely the in vivo conditions of tumor cells than metastatic derived cell lines, we conclude that GnRH analogues have a significant inhibitory effect on cell viability of cells expressing GnRH receptors. In addition, GnRH receptors expressed in tumor prostatic cells seem not discriminate between agonist and antagonist, both analogues activating these receptors. Also, leuprolide and cetrorelix treatments did not influence GnRH receptor expression in our culture system. These differences with pituitary receptors may be explained by differences in affinity, transduction mechanism and molecular context in prostatic tissue.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Hormonais/farmacologia , Antagonistas de Hormônios/farmacologia , Leuprolida/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Ensaio Cometa , Células Epiteliais/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Receptores LHRH/biossíntese , Receptores LHRH/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacosRESUMO
AIM: To assess the role of several genetic factors in combination with an environmental factor as modulators of prostate cancer risk. We focus on allele variants of low-penetrance genes associated with cell control, the detoxification processes and smoking. METHODS: In a case-control study we compared people carrying p53cd72 Pro allele, CYP1A1 M1 allele and GSTM1 null genotypes with their prostate cancer risk. RESULTS: The joint risk for smokers carrying Pro* and M1*, Pro* and GSTM1null or GSTM1 null and CYP1A1 M1* variants was significantly higher (odds ratio [OR]: 13.13, 95% confidence interval [CI]: 2.41-71.36; OR: 3.97, 95% CI: 1.13-13.95 and OR: 6.87, 95% CI: 1.68-27.97, respectively) compared with that for the reference group, and for non-smokers was not significant. OR for combinations among p53cd72, GSTM1 and CYP1A1 M1 in smokers were positively and significantly associated with prostate cancer risk compared with non-smokers and compared with the putative lowest risk group (OR: 8.87, 95% CI: 1.25-62.71). CONCLUSION: Our results suggest that a combination of p53cd72, CYP1A1, GSTM1 alleles and smoking plays a significant role in modified prostate cancer risk on the study population, which means that smokers carrying susceptible genotypes might have a significantly higher risk than those carrying non-susceptible genotypes.
Assuntos
Citocromo P-450 CYP1A1/genética , Genes p53 , Glutationa Transferase/genética , Polimorfismo Genético , Neoplasias da Próstata/genética , Idoso , Intervalos de Confiança , Amplificação de Genes , Variação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Neoplasias da Próstata/epidemiologia , Fatores de Risco , FumarRESUMO
BACKGROUND: GnRH analogs have antiproliferative and/or apoptotic effects on prostate cancer cells. Also, neurotrophin receptors TrkA and p75 have been reported in normal prostate suggesting a role in the gland growth control. In prostate cancer, TrkA receptors seem to be overexpressed and p75 receptors show a decreased expression. These changes in neurotrophin receptors may be related with unbalanced growth in malignant cells. In the present study we investigate the effects of GnRH analogs (leuprolide and cetrorelix) on the expression of TrkA and p75 neurotrophin receptors in primary cultures of human prostate cancer cells. METHODS: Tissue was obtained from radical prostatectomies due to prostate adenocarcinoma. Cells were isolated after sequential enzyme digestion and cultured in defined media. Nerve growth factor (NGF) receptors in untreated cultures were estimated by immunofluorescence. Cultures were treated with leuprolide (agonist) or cetrorelix (antagonist) and expression of TrkA and p75 receptors were evaluated by semi quantitative RT-PCR (polymerase chain reaction) and western blot. Cell proliferation was estimated by MTT method and apoptosis through COMET assay. RESULTS: Both leuprolide and cetrorelix induced a significant increase in p75 receptor gene and protein expression at a concentration that induce apoptosis and decrease proliferation. TrkA receptors showed no changes in presence of GnRH analogs. CONCLUSIONS: GnRH analogs, leuprolide, and cetrorelix, change the ratio between neurotrophin receptors TrkA and p75 by increasing gene and protein expression of p75 receptor. Considering that TrkA receptor is related with proliferation and p75 with apoptosis, we suggest that our findings may explain, in part, the effect of GnRH analogs on prostate cancer growth.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Hormonais/farmacologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Leuprolida/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptor trkA/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Células Epiteliais/patologia , Formazans/química , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor de Fator de Crescimento Neural , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio/químicaRESUMO
BACKGROUND: Prostate cancer (PCa) is one of the most common male cancers, but the burden of this disease shows remarkable worldwide variation. The role of susceptibility low penetrance genes and environmental factors in the etiology of (PCa) is unclear, but may involve, in some cases, multiple alleles at multiple loci and environmental factors. STUDY OBJECTIVES: To assess whether CYP1A1, GSTM1, GSTT1 susceptibility genotypes, smoking status and alcohol consumption factors contribute to PCa risk, gene-gene and gene-environment interactions were analyzed. DESIGN AND PARTICIPANTS: We explored interactions on a multiplicative scale conducting a population-based case-control and a case-only study on 103 incident PCa patients and 132 unrelated controls. MAIN RESULTS: The interaction odds ratios (IOR) for PCa risk were increased in men who had both susceptibility genotypes GST (M1; T1) null and CYP1A1-M1* in a case-control and case-only design (IOR(cc): 1.11; 95% CI: 0.12-10.02; IOR(cc): 6.23; 95%, CI: 0.51-75.89; IOR(co): 2.80; 95% CI: 0.44-17.45 and IORco: 2.65; 95%, CI: 0.30-25.40). No clear evidence for interaction on a multiplicative scale between smoking status, alcohol consumption and genetic polymorphisms in PCa risk was observed. CONCLUSIONS: Our findings suggest that the interaction between genetic polymorphisms in GST (T1; M1) and CYP1A1-M1* would play a significant role as a modifying factor on PCa risk in Chilean people. However, these preliminary exploratory results should be confirmed in a larger study.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Predisposição Genética para Doença , Neoplasias da Próstata/complicações , Fumar/efeitos adversos , Estudos de Casos e Controles , Citocromo P-450 CYP1A1/genética , Glutationa Transferase/genética , Humanos , Masculino , Polimorfismo Genético , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/genética , Fatores de Risco , Estados Unidos/epidemiologiaRESUMO
We investigate the proliferative activity, prostatic specific antigen (PSA) secretion, morphology and androgen response of human prostate tumour epithelial cells co-cultured with stromal cells in a bicameral system. Stromal and epithelial cells were isolated from prostate adenocarcinoma by enzyme digestion and cultured in defined media. Immunocytochemistry for prostate carcinoma tumour antigen (PCTA-1) was performed for culture purity evaluation. Also, the morphology of the epithelial cells in co-culture was evaluated by electron microscopy. PSA was determined by microparticle enzyme immunoassay (MEIA) automatized protocol and the proliferation was evaluated by a commercial spectrophotometric kit, based on formazan salt formation. Both cell cultures showed more than 90% of purity. The epithelial cell co-cultures showed marked membrane processes and cell interdigitations. The proliferative activity of the epithelial cells was increased in presence of stromal cells. Also, PSA secretion was significantly increased and maintained for at least 14 days, whereas the androgen response for PSA secretion was evidenced only in co-culture condition. Primary co-cultures of epithelial and stromal cells from human prostate carcinoma are able to maintain, for a prolonged time, proliferative and secretory properties as well hormone response, and represent a valuable tool for cellular and molecular studies on prostate cancer.
Assuntos
Células Epiteliais/imunologia , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/imunologia , Células Estromais/imunologia , Técnicas de Cocultura , Humanos , Masculino , Neoplasias da Próstata/patologiaRESUMO
AIM: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. METHODS: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nmol/L testosterone. The effect of 1 micromol/L flutamide was also evaluated. RESULTS: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. CONCLUSION: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.
Assuntos
Epididimo/enzimologia , Glutationa Peroxidase/metabolismo , Testosterona/metabolismo , Idoso , Antagonistas de Androgênios/farmacologia , Técnicas de Cultura de Células , Flutamida/farmacologia , Glutationa Peroxidase/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The role of susceptibility low penetrance genes and environmental factors in the etiology of prostate cancer (PCa) is unclear, but may involve in some cases multiple alleles at multiple loci. AIM: To evaluate the association of gene-gene and gene-environment interactions with PCa. PATIENTS AND METHODS: One hundred three subjects with biopsy proven PCa were studied, using a case-only design. All were interrogated about smoking habits. Polymorphisms for Glutathione-S-transferase (GS7) and Cytochrome P4501A1 (CYP1A1), were measured in DNA extracted from peripheral lymphocytes, using a restriction fragment length polymorphism analysis. RESULTS: Our findings suggest that gene-gene interactions between GSTT1 and CYP1A1 high risk genotypes were positive modifiers and had a high predictive value for the presence of PCa, compared with non-susceptibility genotypes. The interaction between susceptibility genotypes and smoking did not modify the risk for PCa. CONCLUSIONS: Gene-gene interactions may play a role modulating the susceptibility to PCa in a proportion of affected individuals.
Assuntos
Meio Ambiente , Polimorfismo Genético , Neoplasias da Próstata/genética , Idoso , Idoso de 80 Anos ou mais , Citocromo P-450 CYP1A1/genética , Predisposição Genética para Doença , Genótipo , Glutationa Transferase/genética , Hábitos , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
BACKGROUND: The prostate cancer is a slowly progressing disease that begins decades prior to diagnosis. It has been suggested that there might be differences in susceptibility due to genetic polymorphisms in biotransformation enzyme genes. In the present work, associations between CYP1A1(Msp1), GSTM1(-/-) polymorphisms, and prostate cancer were analyzed in a case-control study. METHODS: Genomic DNA was isolated from peripheral blood samples, collected on EDTA. PCR-RFLP was used to determine simultaneously Msp1 and GSTM1(-/-) polymorphisms. RESULTS: In cancer patients, frequency of m2 variant allele (0.377) and GSTM1(-/-) (0.362) showed statistically significant increases compared to the control group (0.262 and 0.227, respectively). The estimate relative risks (OR) were higher for individuals carrying combined CYP1A1 and GSTM1 rare genotypes, in relation to individuals carrying CYP1A1 or GSTM1 alone. Multivariate logistic regression analysis including confounding factors (age, digital examination, and PSA antigen) showed even higher risk for individuals carrying m2m2 genotype (OR = 3.99; 95% CI, 1.27-12.54), GST(-/-) genotype (OR = 2.75; 95% CI, 1.31-5.79), and m2m2/GST(-) genotype (OR = 16.63; 95% CI, 1.67-165.48). CONCLUSIONS: Taken together, these findings suggest that Chilean people carrying single or combined GSTM1 and CYP1A1 polymorphisms are more susceptible to prostate cancer.