RESUMEN

The highly similar aldehyde dehydrogenase isozymes (ALDH1A1 and ALDH2) have been implicated in the metabolism of toxic biogenic aldehydes such as 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 4-hydroxy-2E-nonenal. We report the down-regulation of ALDH1A1 mRNA found in substantia nigra tissue of human Parkinson's disease (PD) samples using the Genome-Wide SpliceArray(™) (GWSA(™)) technology. Since DOPAL can rapidly inactivate ALDH1A1 in vitro, we set up a DOPAL-induced ALDH1A1 inactivation assay and used this assay to demonstrate that Alda-1, a compound originally identified as an activator of ALDH2, can also activate ALDH1A1. We carried out a virtual screening of 19,943 compounds and the top 21 hits from this screen were tested in the DOPAL inactivation assay with ALDH1A1 which led to identification of an activator as well as two inhibitors among these hits. These findings represent an attractive starting point for developing higher potency activator compounds that may have utility in restoring the metabolism of DOPAL in PD.

Asunto(s)

Aldehído Deshidrogenasa/genética , Benzamidas/farmacología , Benzodioxoles/farmacología , Enfermedad de Parkinson/enzimología , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Ácido 3,4-Dihidroxifenilacético/metabolismo , Ácido 3,4-Dihidroxifenilacético/farmacología , Aldehído Deshidrogenasa/química , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Aldehído Deshidrogenasa Mitocondrial , Benzamidas/metabolismo , Benzodioxoles/metabolismo , Sitios de Unión , Estudios de Casos y Controles , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Enfermedad de Parkinson/genética , Retinal-Deshidrogenasa , Bibliotecas de Moléculas Pequeñas/farmacología , Sustancia Negra/enzimología , Interfaz Usuario-Computador

RESUMEN

Oxidative stress is known to be one of the major factors underlying Parkinson's disease (PD). One of the consequences of oxidative stress is lipid peroxidation. A toxic product of lipid peroxidation, (±)-4-hydroxy-2E-nonenal (HNE) leads to membrane disruption and formation of HNE-protein adducts and such adducts have been detected in PD brain tissues. Aldehyde dehydrogenases (ALDHs) are involved in metabolizing HNE and other endogenous aldehydes. Interestingly, the cytosolic aldehyde dehydrogenase 1A1 (ALDH1A1) has been reported to be down-regulated in brain tissues affected in PD which could result in enhancement of HNE toxicity. We sought to first establish the role of ALDH1A1 in mediating HNE toxicity in PC12 cells by overexpressing ALDH1A1 and by using disulfiram, an ALDH inhibitor. Overexpression and inhibition of ALDH1A1 activity resulted in reduced and increased HNE toxicity, respectively. We then established conditions for detecting HNE-protein adducts following HNE treatment and showed that overexpression and inhibition of ALDH activity resulted in reduced and increased formation of HNE-protein adducts, respectively. We also show that 6-methyl-2-(phenylazo)-3-pyridinol, previously identified as an activator of ALDH1A1, can protect PC12 cells against HNE-mediated toxicity and can cause a small but significant decrease in levels of HNE-protein adducts. Our results should encourage identification of more potent ALDH activators and their testing in the PC12-HNE model. Such cytoprotective compounds could then be tested for their neuroprotective activity in in vivo models of oxidative stress-induced PD.

Asunto(s)

Aldehídos/toxicidad , Aductos de ADN/metabolismo , Enfermedad de Parkinson/metabolismo , Retinal-Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Aldehídos/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Aductos de ADN/genética , Disulfiram/farmacología , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Células PC12 , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/patología , Ratas , Retinal-Deshidrogenasa/antagonistas & inhibidores , Retinal-Deshidrogenasa/genética

RESUMEN

Hypoxia inducible factor-1 (HIF-1) is a master regulator of cellular adaptation to oxygen deprivation and activates transcription of genes involved in tumor metabolism, angiogenesis, invasion and metastasis, all of which are implicated in cancer progression. Several domains of HIF-1alpha mediate protein-protein interaction, which is essential for the formation of the active heterodimer with HIF-1beta. Targeting specific domains of HIF-1alpha might lead to the identification of more selective inhibitors. HIF-1alpha and HIF-1beta contain two Per-Arnt-Sim (PAS) domains, A and B, both of which appear to be important for heterodimer formation. In an attempt to identify small molecule inhibitors of the PAS-A domain of HIF-1 we expressed proteins containing amino acids 86-165 of HIF-1alpha and amino acids 159-240 of HIF-1beta fused to a His or FLAG tag, respectively. Expressed proteins retained functional activity as indicated by in vitro immunoprecipitation experiments and activation of luciferase expression in a mammalian two-hybrid system. Interestingly, over-expression of HIF-1alpha-PAS-A domain was sufficient to abrogate hypoxic induction of HIF-1-dependent luciferase expression, supporting its potential role as drug target. An ELISA based on the interaction between FLAG-HIF-1beta-PAS-A and HIF-1alpha-PAS-A-His was developed and used to screen libraries of synthetic compounds. NSC 50352 specifically inhibited PAS-A-dependent interaction between HIF-1alpha and HIF-1beta, but not the interaction mediated by unrelated domains. However, NSC 50352 was devoid of activity in cell-based assays. Our results provide proof-of-principle that the PAS-A domain of HIF-1alpha is a valid target for development of small molecule inhibitors.

Asunto(s)

Antineoplásicos/administración & dosificación , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Sistemas de Liberación de Medicamentos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Antineoplásicos/farmacología , Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Genes Reporteros , Secuencias Hélice-Asa-Hélice , Humanos , Factor 1 Inducible por Hipoxia/química , Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Luciferasas , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Elementos de Respuesta/genética , Transcripción Genética , Transfección

RESUMEN

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , ADN de Neoplasias/metabolismo , Equinomicina/farmacología , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , ADN de Neoplasias/química , Ensayo de Inmunoadsorción Enzimática , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/biosíntesis , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Luciferasas/antagonistas & inhibidores , Luciferasas/biosíntesis , Luciferasas/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

Here we report that B16F10 murine melanoma cells mimic endothelial cell behavior and the angiogenic process in vitro and in vivo. Cord formation in vitro by tumor cells is stimulated by hypoxia and vascular endothelial growth factor (VEGF) and inhibited by antibodies against VEGF and the VEGF KDR receptor (VEGF receptor 2). We define regulation of tumor cell-derived vascular space formation by these vasoactive compounds as "vasocrine" stimulation. ICRF 159 (Razoxane; NSC 129943) prevents tumor cell but not endothelial cell cord formation in vitro, and the antiangiogenic drug TNP-470 (NSC 642492) inhibits endothelial but not tumor cell cord formation in vitro. Both drugs inhibit formation of blood-filled vascular spaces in vivo. These results bear on the anticipated action of ICRF 159 in human clinical trials and novel strategies for targeting tumor blood supplies.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/patología , Animales , Antineoplásicos/farmacología , División Celular/fisiología , Hipoxia de la Célula/fisiología , Ciclohexanos , Diseño de Fármacos , Factores de Crecimiento Endotelial/biosíntesis , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Linfocinas/biosíntesis , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , O-(Cloroacetilcarbamoil) Fumagilol , Razoxano/farmacología , Sesquiterpenos/farmacología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Factores de Crecimiento Endotelial Vascular

RESUMEN

Esophagin is a member of the small proline-rich protein family of cell envelope precursor proteins, which are expressed during squamous cell differentiation. Esophagin is expressed at high levels in normal esophageal epithelium, but its expression is absent from esophageal squamous cell carcinomas and adenocarcinomas. Moreover, loss of esophagin expression is present in areas of dysplasia or normal mucosa adjacent to carcinomas, suggesting that absence of esophagin may constitute a harbinger of early esophageal malignant transformation. A greater understanding of transcriptional control of esophagin may provide valuable insights into esophageal malignancy. Therefore, this study was undertaken in order to isolate and carry out initial characterization of a functional promoter for esophagin. A genomic clone containing esophagin was isolated and sequenced, including 2.7 kb of the esophagin promoter region. Esophagin expression was studied in response to various treatments of primary cultured human esophageal epithelial cells and squamous cell carcinoma cell lines. Calcium was the strongest inducer of the endogenous esophagin promoter, with induction occurring at 12-72 hours. In primary cultured esophageal epithelial cells, a region spanning 116 bp upstream of the transcriptional start site to 45 bp downstream was sufficient to direct low, basal, in vitro esophagin expression. However, responsiveness of primary esophageal cells to calcium required inclusion of promoter elements 1688 bp upstream of the transcriptional start site. Site-directed mutagenesis studies suggested a putative role for C/EBP-beta, OCT-1, and OCT-3 transcription factor binding sites in the minimal promoter region. In conjunction with published human in vivo studies, these data support the hypothesis that esophagin is a biomarker of esophageal squamous cell differentiation and provide an in vitro model to evaluate regulatory factors involved in this differentiation process.

Asunto(s)

Esófago/metabolismo , Péptidos/genética , Regiones Promotoras Genéticas , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Esófago/crecimiento & desarrollo , Regulación de la Expresión Génica , Humanos , Péptidos/metabolismo , Dominios Proteicos Ricos en Prolina , Eliminación de Secuencia