RESUMEN
Histone deacetetylases (HDACs) are a group of corepressors of transcriptional activators and their levels of expression are potentially dysregulated in prostate cancer. Certain inhibitors of histone deacetylases show anti-tumor activity in prostate cancer cell lines. Here, we systemically studied the expression of HDACs in human prostate cancer and the suppression of prostate cancer growth and invasion by HDAC inhibitor SAHA. HDAC1-5 showed increased expression using a combination of DNA microarray, in-situ hybridization, and immunohistochemistry in benign and malignant human prostate tissue as well as RT-PCR and Western blot analysis on various PCa cell lines. Importantly, HDAC inhibitor SAHA suppressed, in particular, prostate cancer cell growth and invasion determined using cell proliferation and Matrigel invasion assays. The findings of this study show that the expression of HDACs and their associated corepressors are increased in prostate cancer in humans and HDAC inhibitor SAHA could serve as a potential therapeutic agent in prostate cancer in addition to anti-androgens.
RESUMEN
A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease.
Asunto(s)
Factor de Unión 1 al Potenciador Linfoide/biosíntesis , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/biosíntesis , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Humanos , Factor de Unión 1 al Potenciador Linfoide/genética , Masculino , Microscopía Fluorescente , Invasividad Neoplásica , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Increased androgen receptor (AR) expression and activity are pivotal for androgen-independent (AI) prostate cancer (PC) progression and resistance to androgen-deprivation therapy. We show that a novel transcriptional repressor complex that binds a specific sequence (repressor element) in the AR gene 5'-untranslated region contains Pur alpha and hnRNP-K. Pur alpha expression, its nuclear localization, and its AR promoter association, as determined by chromatin immunoprecipitation analysis, were found to be significantly diminished in AI-LNCaP cells and in hormone-refractory human PCs. Transfection of AI cells with a plasmid that restored Pur alpha expression reduced AR at the transcription and protein levels. Pur alpha knockdown in androgen-dependent cells yielded higher AR and reduced p21, a gene previously shown to be under negative control of AR. These changes were linked to increased proliferation in androgen-depleted conditions. Treatment of AI cells with histone deacetylase and DNA methylation inhibitors restored Pur alpha protein and binding to the AR repressor element. This correlated with decreased AR mRNA and protein levels and inhibition of cell growth. Pur alpha is therefore a key repressor of AR transcription and its loss from the transcriptional repressor complex is a determinant of AR overexpression and AI progression of PC. The success in restoring Pur alpha and the repressor complex function by pharmacologic intervention opens a promising new therapeutic approach for advanced PC.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Regiones no Traducidas 5'/genética , Línea Celular Tumoral , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Genes Reporteros , Humanos , Inmunohistoquímica , Luciferasas/genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Represoras/genética , Factores de Transcripción/metabolismoRESUMEN
Epidemiological surveys indicate that intake of cruciferous vegetables is inversely related to prostate cancer incidence, although the responsible dietary factors have not been identified. Our studies demonstrated that exposure of human prostate cancer cells in culture to the N-acetylcysteine (NAC) conjugate of phenethyl isothiocyanate (PEITC-NAC), the major metabolite of PEITC that is abundant in watercress, inhibited proliferation and tumorigenesis. The PEITC-NAC is known to mediate cytoprotection at initiation of carcinogenesis. The relevance of PEITC-NAC in diets on the growth of prostate tumor cells has been evaluated in immunodeficient mice with xenografted tumors of human prostate cancer PC-3 cells. The daily PEITC-NAC (8 micromol/g) supplemented diet group showed a significant reduction in tumor size in 100% of the mice during the 9-week treatment period. Tumor weight at autopsy was reduced by 50% compared with mice on the diet without PEITC-NAC (P = 0.05). Mitosis and in vivo 5-bromo-2'-deoxyuridine labeled proliferating cells were reduced in these tumors. The PEITC-NAC diet up-regulated the inhibitors of cyclin-dependent kinases p21WAF-1/Cip-1 and p27Kip1, and reduced the expression of cyclins D and E, indicating they were potential molecular targets. As a result, phosphorylated Rb was significantly decreased and the G1- to S-phase transition retarded. The treated tumors also showed a significant increase in apoptosis as determined by in situ end-labeling, and by poly ADP-ribose polymerase cleavage. This study demonstrates the first in vivo evidence of dietary PEITC-NAC inhibiting tumorigenesis of prostate cancer cells. PEITC-NAC may prevent initiation of carcinogenesis and modulate the post-initiation phase by targeting cell cycle regulators and apoptosis induction.
Asunto(s)
Isotiocianatos/metabolismo , Verduras/metabolismo , Acetilcisteína/química , Animales , Anticarcinógenos/uso terapéutico , Apoptosis , Western Blotting , Bromodesoxiuridina/farmacología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , División Celular , Línea Celular Tumoral , Separación Celular , Colorantes/farmacología , Ciclina D , Ciclina E/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Citometría de Flujo , Fase G1 , Humanos , Isotiocianatos/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitosis , Trasplante de Neoplasias , Neoplasias/metabolismo , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Fase S , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Epidemiological studies recently concluded that consumption of cruciferous vegetables such as broccoli, cabbage, and cauliflower, etc. is inversely related to prostate cancer risk, although the mechanism of prevention and the responsible phytochemicals are unknown. Since clinically significant prostate cancer eventually can grow independent of androgen, the association of the growth and tumorigenesis of such prostate cancer cells with sulforaphane (SFN) which is a predominant isothiocyanate in cruciferous vegetables, investigated. These vegetables contain high concentrations of glucosinolate glucoraphanin, which yield sulforaphane when hydrolyzed by the plant enzyme myrosinase. This study showed that exposure of human androgen-independent DU-145 prostate cancer cells to SFN resulted in the inhibition of growth and tumorigenesis, as revealed by a reduction in cell density, DNA synthesis, and clonogenesis. Analyses of the mechanism revealed that SFN mediated cell cycle arrest by modulating the expression and functions of cell cycle regulators. SFN induced signals that inhibited the activity of cyclin-dependent kinase cdk4 with an up-stream induction of cdk inhibitor p21WAF-1/Cip-1, and reduced cyclin D1. The inhibition of cdk kinase activity could be affected with <1 micro M SFN within 24 h. As a result, phosphorylation of Rb proteins, which activates the transition from G1- to S-phase, was significantly decreased and the cell cycle progression retarded. SFN also down-regulated the expression of bcl-2, a suppressor of apoptosis, and activated caspases to execute apoptosis in the prostate cancer cells. The regulators of cell cycle have thus been revealed as targets of sulforaphane for growth arrest and apoptosis induction. The potential of SFN, as an active dietary factor to inhibit initiation and post-initiation of prostate cancer carcinogenesis is discussed.
Asunto(s)
Anticarcinógenos/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas , Tiocianatos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Fase G1/efectos de los fármacos , Humanos , Etiquetado Corte-Fin in Situ , Isotiocianatos , Masculino , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína de Retinoblastoma/metabolismo , Fase S/efectos de los fármacos , Sulfóxidos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
3-Iodoacetamido benzoyl ethyl ester (3-IAABE) is a new compound synthesized in our laboratory. The primary action of 3-IAABE is to inhibit microtubule assembly by interacting with -SH groups on tubulin. In contrast to other known microtubule disrupters, 3-IAABE caused a double blockade in the cell cycle at G(1)-S transition and in M phase. The blockade was determined by cell cycle analysis and chromosome distribution. Kinase activities of cyclin E and cyclin-dependent kinase 2 responsible for the G(1)-S transition were increased, as were the activities of mitotic cyclin B and cdc2. 3-IAABE treatment also increased p53 expression and dephosphorylated (or activated) retinoblastoma protein. Investigation of the signal transduction pathway showed that 3-IAABE induced bcl-2 phosphorylation, followed by activation of caspase-9, -3, and -6, but not caspase-8. DNA fragmentation factor and poly(ADP-ribose) polymerase, the downstream substrates of caspase-3 and -6, were cleaved after 3 h of exposure to 3-IAABE, followed by DNA fragmentation. Pretreatment of the cells with inhibitors of caspase-9, -3, or -6, respectively, inhibited the cleavage of DNA fragmentation factor and poly(ADP-ribose) polymerase and thus inhibited the onset of apoptosis. 3-IAABE showed antitumor activities in the panel of 60 National Cancer Institute human tumor cell lines with total growth inhibition in the range of 0.22-4.3 micro M for solid tumor lines and 0.025-0.22 micro M for leukemia/lymphoma cell lines. The 3-IAABU total growth inhibition of phytohemagglutinin-stimulated healthy human lymphocytes was 450-fold greater than that of leukemic cells. 3-IAABE significantly inhibited the growth of human hepatocarcinoma (BEL-7402) in nude mice by 72% in tumor volume, more strongly than did vincristine (43 percent inhibition). Besides being a novel lead for the design of new anticancer tubulin ligands, the activity of 3-IAABE in the cell cycle may also help us to understand the molecular pharmacology of microtubule-active drugs.