RESUMEN
The Aerogel Cherenkov Detector for Cygnus (ACD/C) is a time-dependent, x-ray spectral detector that uses SiO2 aerogels spanning an index of refraction (n = 1.02-1.07) corresponding to a 1.1-2.3 MeV x-ray energy threshold. The ACD/C was developed for pulsed power x-ray sources like Cygnus located at the Nevada National Site and Mercury located at the Naval Research Laboratory (NRL). Aerogels sit between the measurement capabilities of gas (>2 MeV) and solids such as fused silica (>0.3 MeV). The detector uses an aluminum converter to Compton scatter incoming x-rays and create relativistic electrons, which produce Cherenkov light in an aerogel or a fused silica medium. The ACD/C was fielded at the NRL when Mercury was tuned to produce up to 4.8 MeV endpoint bremsstrahlung. Despite a high radiation and electromagnetic interference background, the ACD/C was able to achieve high signal over noise across five aerogel densities and fused silica, including a signal to noise for a 1.1 MeV aerogel threshold. Previous experiments at Cygnus observed a signal that was comparable to the noise (1×) at the same threshold. The ACD/C observed time-resolved rise and fall times for different energy thresholds of the photon spectrum. Monte Carlo simulations of the ACD/C's aerogel response curves were folded with a simulation of Mercury's photon energy spectrum and agree within the error to the observed result.
RESUMEN
We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome ß5 and ß1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF(2α)), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF(2α) and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as a potential agent in cancer chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Autofagia , Bibencilos/farmacología , Éteres Fenílicos/farmacología , Neoplasias de la Próstata/patología , Inhibidores de Proteasoma/farmacología , Antineoplásicos/química , Bibencilos/química , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Masculino , Éteres Fenílicos/química , Neoplasias de la Próstata/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/química , Transducción de SeñalRESUMEN
BACKGROUND: Surgical excision of papillary breast lesions with atypia diagnosed using core needle biopsy (CNB) has been accepted; however, the management of benign papillary lesions (without atypia) has been controversial. The purpose of this study was to evaluate the surgical outcome of nonmalignant papillary lesions diagnosed by ultrasound-guided 14-gauge CNB, and to establish clear guidelines on management of these lesions. METHODS: We retrospectively identified 268 nonmalignant papillary breast lesions, including 203 benign lesions and 65 atypical lesions, diagnosed by CNB and subsequently surgically excised in 250 women at our institution between July 2004 and October 2010. For each lesion, medical records and radiologic and pathologic reports were reviewed and coded. We compared the histological upgrade among the collected variables. RESULTS: On histological examination after surgical excision, 15.4% atypical papillary lesions and 5.9% benign lesions were upgraded to malignant, and 20.2% benign lesions were upgraded to atypical. Atypia (P = 0.015) was significantly associated with malignant upgrade at excision. No clinical or radiologic variable was helpful in predicting the possibility of histological upgrade of CNB-diagnosed nonmalignant papillary lesions. CONCLUSIONS: Nonmalignant papillary lesions diagnosed with CNB showed an unacceptable pathological upgrade rate after excision. Therefore, surgical excision should be performed for all papillary lesions of the breast for definitive diagnosis.
Asunto(s)
Biopsia con Aguja Gruesa , Neoplasias de la Mama/cirugía , Carcinoma Papilar/cirugía , Biopsia Guiada por Imagen , Papiloma/diagnóstico , Papiloma/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/patología , Femenino , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Somatic genetic and epigenetic alterations have been suggested to be crucially involved in development and progression of cancers including prostate cancer (PCa). Epigenetic alterations such as chemical modification of chromatin associated proteins and DNA methylation can largely affect gene expression that may be important for early normal organ development, and also produces favorable conditions for cancer cell formation, growth, and survival. Aberrant DNA methylation (hyper- or hypo-methylation) may lead to chromosomal instability, and transcriptional gene silencing for tumor suppressors or overexpression for oncogenes. Vitamin Bs play important roles in one carbon metabolism that provides critical metabolites for DNA methylation, DNA repair and nucleic acid synthesis. Nutrition uptake and circulating levels of these vitamin Bs as well as genetic polymorphisms of related key enzymes in the one carbon metabolism pathway may govern bioavailability of the metabolites, and therefore to affect the phenotypic changes (e.g., cellular malignancy) via genetic and epigenetic alterations. This article will summarize recent new findings from laboratory, epidemiological or clinical trial studies regarding influence of vitamin B and one carbon metabolism on PCa development or progression.
Asunto(s)
Carbono/metabolismo , Neoplasias de la Próstata/metabolismo , Complejo Vitamínico B/química , Animales , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Complejo Vitamínico B/metabolismoRESUMEN
Androgen deprivation therapy has been the major treatment for advanced prostate cancer (PCa) and has shown to prolong life. However, remissions are temporary and patients almost inevitably progress to become castration-resistant prostate cancer (CRPC). CRPC is almost incurable even when treated with docetaxel that may have a slight life prolonging effect on CRPC patients. Interestingly, most of CRPC still express androgen receptor (AR) and depend on the AR for growth. Recently it has been suggested that AR may act as a tumor suppressor in normal prostatic epithelial cells, while in PCa cells AR becomes oncogenic, even under androgen deprivation states. The mechanisms for the latter are still under intensive investigations. A number of studies showed that, in fact, AR signaling is increased under an androgen-depleted environment. The mechanisms suggested in these studies including AR mutations, AR overexpression by gene amplification and other mechanisms that allow activation by low androgen levels or by other endogenous steroids, increased local de novo synthesis of androgens will be discussed. Moreover, developments and tests in clinical trials in CRPC of a number of novel agents interrupting AR signaling mediated PCa growth will also be discussed.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Andrógenos/fisiología , Ciclo Celular , Resistencia a Antineoplásicos , Humanos , Masculino , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/fisiopatología , Empalme del ARN , Receptores Androgénicos/genética , Receptores Androgénicos/fisiologíaRESUMEN
Epidermal growth faxtor receptor (EGFR)-vIII mutant has been demonstrated to over-express as prostatic neoplasms progressed from intraepithelial changes to metastatic disease. In this study, we transfected the EGFRvIII expression vector into an immortalized normal prostate epithelium cell line RWPE-1 and established stable transfectants. The cell growth, glandular morphogenesis, cell motility, and soft-agar colony formation efficiency were then studied. The results showed that EGFR-vIII mutation increased the RWPE1 cell motility and clone formation efficiency, while it had no significant effect on the cell growth when compared to non-transfected as well as mock transfected RWPE-1 cells. Moreover, EGFR-vIII changed the RWPE1 acinar morphogenesis. Further study showed that these effects of EGFR-vIII mutation may be related to down-regulation of E-cadherin and up-regulation of beta-catenin.
Asunto(s)
Línea Celular , Movimiento Celular/genética , Proliferación Celular , Receptores ErbB/fisiología , Próstata/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/genética , Transformación Celular Neoplásica/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Humanos , Masculino , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Transfección , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Chemoprevention is presumably one of most effective means to combat prostate cancer (PCa). Patients usually require more than a decade to develop a clinically significant Pca, therefore, an ideal target for chemoprevention. This review will focus on recent findings of a group of naturally occurring chemicals, carotenoids, for potential use in reducing PCa risk.
Asunto(s)
Carotenoides/uso terapéutico , Neoplasias de la Próstata/prevención & control , Antioxidantes/química , Antioxidantes/uso terapéutico , Carotenoides/química , Humanos , Masculino , Factores de RiesgoRESUMEN
We have recently identified ZNF185 as a gene that is downregulated in prostate cancer (PCa), in part via epigenetic alteration, and maybe associated with disease progression. In this study, we cloned the ZNF185 cDNA from normal human prostate tissues and investigated its biological function. We show that ZNF185 is a novel actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein that localizes to F-actin structures, and is enriched at focal adhesions. We find that the NH(2)-terminal region, which we designate the actin-targeting domain, facilitates ZNF185 binding to actin in vitro and is both necessary and sufficient to mediate actin-cytoskeleton targeting of ZNF185, whereas the LIM domain, which is localized in the COOH-terminus is dispensable for this phenomenon. Interestingly, ectopic expression of full-length ZNF185, but not a mutant lacking the actin-targeting domain, could suppress proliferation and anchorage-independent growth of PCa cells. Together, our data suggest that ZNF185 may function as a tumor-suppressor protein by associating with the actin-cytoskeleton.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/metabolismo , Neoplasias de la Próstata/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Línea Celular Tumoral , Proteínas del Citoesqueleto , ADN Complementario , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Proteínas con Dominio LIM , Masculino , Datos de Secuencia Molecular , Neoplasias de la Próstata/patología , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Although heat-shock protein 70 (Hsp70) has been considered an intracellular protein, we report that Hsp70 is secreted under normal cell culture conditions by human prostate cell lines, LAPC-4, PC-3, CWR-22, RWPE-1 and -2, LNCaP, and TRAMP (transgenic adenocarcinoma mouse prostate)-C2. We found that the secretion can be enhanced by transfection with cDNA encoding for Hsp70. To verify that the Hsp70 detected in the supernatant was not secondary to cell leakage, C2 cells were cotransfected with cytoplasmic Renilla luciferase as a reporter. High levels of activities were noted in the cell extracts, while no enzyme activities were detected in the supernatants. To verify that forced oversecretion of Hsp70 could protect against tumour growth, mice were injected with C2 cells transfected with an Hsp70 DNA construct and challenged with live tumour cells. Mice injected with cells transfected with the Hsp70 DNA construct demonstrated a significantly decreased rate of tumour growth compared to those injected with empty vector. In addition, a difference in survival rate as defined by a surrogate end point was noted between the two groups. In a second experiment, we developed a cell line that stably overexpressed Hsp70. Mice injected with these cells also demonstrated a significant decrease in tumour growth and significantly increased survival.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/fisiopatología , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/fisiopatología , Adenocarcinoma/prevención & control , Animales , Western Blotting , Quimioprevención , Citoplasma , ADN Complementario , Proteínas HSP70 de Choque Térmico/farmacología , Humanos , Masculino , Ratones , Ratones Transgénicos , Neoplasias de la Próstata/prevención & control , Transfección , Células Tumorales CultivadasRESUMEN
We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.
Asunto(s)
Expresión Génica/efectos de los fármacos , Radioisótopos de Yodo/uso terapéutico , Neoplasias de la Próstata/radioterapia , Simportadores/genética , Tretinoina/farmacología , Adenocarcinoma/metabolismo , Adenocarcinoma/radioterapia , Northern Blotting , Western Blotting , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Terapia Genética , Humanos , Yoduros/metabolismo , Cinética , Masculino , Regiones Promotoras Genéticas , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of thyroid-specific expression of the sodium iodide symporter (NIS). Here we explore the efficacy of a novel form of gene therapy using adenovirus-mediated in vivo NIS gene transfer followed by (131)I administration for treatment of prostate cancer. Prostate cancer xenografts in nude mice injected with an adenovirus carrying the NIS gene linked to the cytomegalovirus (CMV) promoter revealed highly active uptake of radioiodine. Following administration of 3 mCi of (131)I, we observed an average tumor volume reduction of 84 +/- 12%. These results show for the first time that in vivo NIS gene delivery into non-thyroidal tumors is capable of inducing accumulation of therapeutically effective radioiodine doses and might therefore represent an effective and potentially curative therapy for prostate cancer.
Asunto(s)
Adenocarcinoma/terapia , Terapia Genética/métodos , Radioisótopos de Yodo/uso terapéutico , Neoplasias de la Próstata/terapia , Simportadores/genética , Transfección/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/radioterapia , Adenoviridae/genética , Animales , Fusión Artificial Génica/métodos , Western Blotting/métodos , Línea Celular , Vectores Genéticos/administración & dosificación , Humanos , Inmunohistoquímica/métodos , Radioisótopos de Yodo/metabolismo , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , Simportadores/metabolismo , Trasplante HeterólogoRESUMEN
A number of reports have shown that the polyphenolic flavonoid silymarin (SM) is an effective anticancer agent. Agents with novel mechanisms of blocking androgen receptor (AR) function may be useful for prostate cancer prevention and therapy. Previous studies showed that silibinin (SB), the major active component of SM, could inhibit cell proliferation of a human prostate cancer cell line, LNCaP, by arresting the cell cycle at the G(1) phase without causing cell death. This study further delineates the potential molecular mechanism by which SM and SB exhibit antiproliferative effects on androgen-responsive prostate cancer cells by inhibiting function of the AR. We observed that SM and SB inhibited androgen-stimulated cell proliferation as well as androgen-stimulated secretion of both prostate-specific antigen (PSA) and human glandular kallikrein (hK2). Additionally, for the first time, we show that an immunophilin, FKBP51, is androgen regulated and that this up-regulation is suppressed by SM and SB. We further demonstrate that transactivation activity of the AR was diminished by SM and SB using gene transfer of PSA promoter and hK2 androgen-responsive element constructs. However, expression and steroid-binding ability of total AR were not affected by SM in western blotting and ligand-binding assays. Intriguingly, we found that nuclear AR levels are significantly reduced by SM and SB in the presence of androgens using western blotting assay and immunocytochemical staining. This study provides a new insight into how SM and SB negatively modulate androgen action in prostate cancer cells.
Asunto(s)
Antagonistas de Receptores Androgénicos , Neoplasias de la Próstata/metabolismo , Silimarina/farmacología , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Masculino , Nandrolona/análogos & derivados , Nandrolona/farmacología , Antígeno Prostático Específico/biosíntesis , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Receptores Androgénicos/fisiología , Proteínas de Unión a Tacrolimus/biosíntesis , Proteínas de Unión a Tacrolimus/genética , Congéneres de la Testosterona/farmacología , Calicreínas de Tejido/biosíntesis , Calicreínas de Tejido/genética , Activación Transcripcional/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
There is some epidemiological support for a protective influence of omega-3 fatty acids against prostate cancer. We wanted to explore whether omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) can affect androgen receptor function in prostate cancer cells. Our study showed that both DHA and EPA inhibit androgen-stimulated cell growth. Androgenic induction of prostate-specific antigen (PSA) protein was repressed by DHA and EPA in a dose-dependent manner. The mRNA levels of five androgen up-regulated genes, PSA, ornithine decarboxylase, NKX 3.1, immunophilin fkbp 51 and Drg-1, were decreased with DHA treatment in the presence of androgens. Transfection experiments using a DNA vector containing androgen-responsive elements demonstrated that both DHA and EPA could interfere with transactivation activities of the androgen receptor (AR). However, western blot analysis of AR protein showed that DHA and EPA treatments did not change AR expression levels. Interestingly, the proto-oncoprotein c-jun was increased by DHA treatment. A transient transfection found that forced expression of c-jun inhibited AR transactivation activity. Thus, this study found that the inhibitory effects of omega-3 polyunsaturated fatty acids on AR-mediated actions are due, at least in part, to an increase in c-jun protein.
Asunto(s)
Antagonistas de Receptores Androgénicos , Andrógenos/fisiología , División Celular/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Expresión Génica/efectos de los fármacos , Northern Blotting , Western Blotting , División Celular/fisiología , Expresión Génica/fisiología , Humanos , Masculino , ARN Mensajero/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Neuroendocrine (NE) differentiation may be related to the growth and progression of prostate cancer, especially androgen-insensitive tumors. Recently the over-expression of a new anti-apoptosis protein, survivin, has attracted attention for its potential implication in many human cancers. The fact that NE cells in prostate are bcl-2 negative prompted us to investigate if the prostatic NE cells over-express survivin. METHODS: Double immunohistochemical staining and immunofluorescence of chromogranin A (CgA) and survivin were performed in 57 patients with localized prostate cancer who underwent radical prostatectomy. The terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) method was used for apoptosis detection in three prostate cancer specimens with NE differentiation. The relationship between NE differentiation and clinicopathological characteristics, disease progression as well as patient survival, were analyzed retrospectively. RESULTS: It was found that NE cells in both benign and malignant prostate tissues over-expressed the anti-apoptosis protein survivin. While apoptosis was detected in non-NE epithelial cells, all NE cells were negative for apoptosis detection. During the period of follow-up, 17 (63%) of 27 patients with NE differentiation had prostate cancer progression, while 12 (40%) of 30 patients without NE differentiation had systemic prostate cancer progression. 10 (37%) of 27 patients with NE differentiation died from prostate cancer during the period of follow up, while 6 (20%) of 30 patients without NE differentiation died from prostate cancer. However, none of these characteristics reached statistical significance, probably because of the small number of cases enrolled. CONCLUSIONS: This study discovers that all the prostatic NE cells express the new anti-apoptosis protein survivin. This provides a strong molecular basis for the hypothesis that NE cells may endure stressful conditions and escape from apoptosis. While our results suggest a trend of NE differentiation with poorer prognosis, the prognosis implication cannot be concluded due to our small sample size.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/patología , Próstata/citología , Próstata/patología , Neoplasias de la Próstata/patología , Proteínas/análisis , Anciano , Apoptosis/fisiología , Biomarcadores de Tumor , Distribución de Chi-Cuadrado , Cromogranina A , Cromograninas/inmunología , Progresión de la Enfermedad , Humanos , Inmunoglobulina G/inmunología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Proteínas Inhibidoras de la Apoptosis , Masculino , Persona de Mediana Edad , Morfogénesis , Proteínas de Neoplasias , Pronóstico , Prostatectomía , Neoplasias de la Próstata/inmunología , Células Madre/citología , SurvivinRESUMEN
Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.
Asunto(s)
Terapia Genética/métodos , Regiones Promotoras Genéticas , Próstata/metabolismo , Calicreínas de Tejido/biosíntesis , Calicreínas de Tejido/genética , Adenoviridae/genética , Animales , Sitios de Unión , Encéfalo/metabolismo , Citomegalovirus/genética , Relación Dosis-Respuesta a Droga , Elementos de Facilitación Genéticos , Citometría de Flujo , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Hígado/metabolismo , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Modelos Genéticos , Trasplante de Neoplasias , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Fluorescencia , Transcripción Genética , Transducción Genética , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The androgen receptor (AR) is involved in the development and progression of prostate cancer. In order to find new compounds that may present novel mechanisms to attenuate the function of AR, we investigated the effect of a natural flavonoid chemical, quercetin, on androgen action in an androgen-responsive LNCaP prostate cancer cell line. Western blot analysis showed that AR protein expression was inhibited by quercetin in a dose-dependent manner. To demonstrate that the repression effects on AR expression can actually reduce its function, we found that quercetin inhibited the secretion of the prostate-specific, androgen-regulated tumor markers, PSA and hK2. The mRNA levels of androgen-regulated genes such as PSA, NKX3.1 as well as ornithine decarboxylase (ODC) were down-regulated by quercetin. Transient transfections further showed that quercetin inhibited AR-mediated PSA expression at the transcription level. Finally, it was demonstrated that quercetin could repress the expression of the AR gene at the transcription level. Our result suggests that quercetin can attenuate the function of AR by repressing its expression and has the potential to become a chemopreventive and/or chemotherapeutic agent for prostate cancer.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Andrógenos/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
Prostate-specific antigen (PSA) is the most useful biomarker for human prostate cancer and may play a role in prostate tumor biology. Androgens, via their receptors, are the major positive regulators of PSA expression. Recently, we showed that thyroid hormone 3,5,3'-L-triiodothyronine (T3) also increases androgen-dependent PSA expression, even though androgen receptor expression is not affected. This report demonstrates for the first time that there is a functional T3-responsive element (TRE) in the 5'-promoter region of the PSA gene. Mutation of this TRE reduced the T3-enhanced androgenic activation of the PSA promoter. Our study provides direct evidence that the PSA gene is regulated by T3 at the transcriptional level.
Asunto(s)
Andrógenos/metabolismo , Antígeno Prostático Específico/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Triyodotironina/farmacología , Northern Blotting , Western Blotting , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Regiones Promotoras Genéticas , Antígeno Prostático Específico/análisis , Neoplasias de la Próstata , ARN Mensajero/análisis , Transfección , Células Tumorales CultivadasRESUMEN
Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.
Asunto(s)
Adenocarcinoma/radioterapia , Proteínas Portadoras/genética , Radioisótopos de Yodo/uso terapéutico , Proteínas de la Membrana/genética , Neoplasias de la Próstata/radioterapia , Simportadores , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Western Blotting , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , Terapia Genética , Humanos , Inmunohistoquímica , Radioisótopos de Yodo/farmacocinética , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Use of the World Wide Web provides an efficient means to disseminate guidelines, but integrating them into the workflow at the point of care remains elusive. We developed a method, ActiveGuidelines (AGL), of integrating web-based guidelines with computer-based patient record (CPR) systems. An ActiveGuideline is an HTML document containing special tags that are interpreted by a CPR as actions (e.g., medication order, test order, referral, patient instructions). In our usage scenario, the CPR automatically displays ActiveGuidelines relevant to the current patient context. After reviewing the guideline, the user selects recommended orders directly from the ActiveGuideline. The selected orders are automatically transmitted to the CPR and executed as regular orders. An ActiveGuideline editor facilitates easy conversion of existing HTML-formatted guidelines into ActiveGuidelines. We believe that integrating patient-specific ActiveGuidelines within a CPR system will improve utilization of clinical guidelines in routine patient care.
Asunto(s)
Internet , Sistemas de Registros Médicos Computarizados/organización & administración , Guías de Práctica Clínica como Asunto , Integración de Sistemas , Sistemas de Computación , Toma de Decisiones Asistida por Computador , Humanos , Hipermedia , Programas InformáticosRESUMEN
Human glandular kallikrein (hK2) is an androgen regulated protein primarily expressed in the prostate and recently identified as a novel prostate cancer marker. A 5 kb 5' flanking region of the hK2 gene was isolated and sequenced to characterize the regulatory mechanisms for the expression of hK2 in the androgen responsive prostate cell line, LNCaP. Using gene transfer, gel shift, and mutagenesis assays we have identified an ARE in the 5' far upstream promoter region of the hK2 gene that is crucial for its regulation in LNCaP cells. This study further demonstrated that the hK2 upstream ARE plays a predominant role in androgenic response. More interestingly, previously identified AREs in the prostate specific antigen promoter and the hK2 proximal promoter exert little activity in LNCaP cells. This study for the first time identifies a unique ARE that alone mediates the function of the androgen receptor in LNCaP cells in a cell dependent manner. This study also examines the activity of this ARE with 1alpha, 25 dihydroxy-vitamin D3 on the expression of the hK2 gene in LNCaP cells.