RESUMEN
In a three-dimensional (3D) representation, each protein molecule displays a specific pattern of chemical and topological features, which are altered during its misfolding and aggregation pathway. Generating a recognizable fingerprint from such features could provide an enticing approach not only to identify these biomolecules but also to gain clues regarding their folding state and the occurrence of pathologically lethal misfolded aggregates. We report here a universal strategy to generate a fluorescent fingerprint from biomolecules by employing the pan-selective molecular recognition feature of a cucurbit[7]uril (CB[7]) macrocyclic receptor. We implemented a direct sensing strategy by covalently tethering CB[7] with a library of fluorescent reporters. When CB[7] recognizes the chemical and geometrical features of a biomolecule, it brings the tethered fluorophore into the vicinity, concomitantly reporting the nature of its binding microenvironment through a change in their optical signature. The photophysical properties of the fluorophores allow a multitude of probing modes, while their structural features provide additional binding diversity, generating a distinct fluorescence fingerprint from the biomolecule. We first used this strategy to rapidly discriminate a diverse range of protein analytes. The macrocyclic sensor was then applied to probe conformational changes in the protein structure and identify the formation of oligomeric and fibrillar species from misfolded proteins. Notably, the sensor system allowed us to differentiate between different self-assembled forms of the disease-specific amyloid-ß (Aß) aggregates and segregated them from other generic amyloid structures with a 100% identification accuracy. Ultimately, this sensor system predicted clinically relevant changes by fingerprinting serum samples from a cohort of pregnant women.
Asunto(s)
Péptidos beta-Amiloides , Hidrocarburos Aromáticos con Puentes , Amiloide , Péptidos beta-Amiloides/química , Hidrocarburos Aromáticos con Puentes/química , Femenino , Colorantes Fluorescentes/química , Compuestos Heterocíclicos con 2 Anillos , Humanos , Imidazoles/química , Imidazolidinas , Compuestos Macrocíclicos , EmbarazoRESUMEN
Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz et al. (2015) J. Antibiot. , DOI: 10.1038/ja.2015.84]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone, and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIα were assessed. GE-2 enhanced DNA cleavage â¼4-fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including (1) the inability to poison a topoisomerase IIα construct that lacks the N-terminal domain, (2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and (3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIα except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIα may play a role in mediating the cytotoxic effects of these fungal metabolites.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Benzaldehídos/farmacología , Ciclohexanonas/farmacología , División del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Hongos/química , Hongos/metabolismo , Metabolismo Secundario , Benzaldehídos/química , Benzaldehídos/metabolismo , Ciclohexanonas/química , Ciclohexanonas/metabolismo , Humanos , Estructura MolecularRESUMEN
Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is an antineoplastic agent that intercalates into DNA and alters topoisomerase II activity. Unfortunately, this compound displays a number of adverse properties. Therefore, to investigate new ellipticine-based compounds for their potential as topoisomerase II-targeted drugs, we synthesized two novel derivatives, N-methyl-5-demethyl ellipticine (ET-1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2). As determined by DNA decatenation and cleavage assays, ET-1 and ET-2 act as catalytic inhibitors of human topoisomerase IIα and are both more potent than the parent compound. Neither compound impairs the ability of the type II enzyme to bind its DNA substrate. Finally, the potency of ET-1 and ET-2 as catalytic inhibitors of topoisomerase IIα appears to be related to their ability to intercalate into the double helix.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Elipticinas/química , Elipticinas/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Antígenos de Neoplasias/metabolismo , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , MetilaciónRESUMEN
ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase II-mediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Histona Acetiltransferasas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , ADN Complementario/metabolismo , Proteínas de Unión al ADN , Genes Reporteros , Células HeLa , Histona Acetiltransferasas/genética , Humanos , Microscopía Fluorescente , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 2/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
A set of solanidine analogs with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.
Asunto(s)
Diosgenina/química , Diosgenina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Acetatos/química , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diosgenina/síntesis química , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Células HeLa , Humanos , Ratones , Neoplasias/patología , Pregnadienodioles/síntesis química , Pregnadienodioles/químicaRESUMEN
Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Encéfalo , Proteínas del Citoesqueleto/genética , Feto , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Genes , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Microscopía Confocal , Datos de Secuencia Molecular , Dominios PDZ , Mapeo de Interacción de Proteínas , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.
Asunto(s)
Proteínas Fúngicas/metabolismo , Kluyveromyces/genética , Recombinación Genética , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas Fúngicas/genética , Técnicas de Inactivación de Genes , Kluyveromyces/enzimología , Kluyveromyces/crecimiento & desarrollo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Eliminación de Secuencia , Telomerasa/genética , Telómero/genéticaRESUMEN
The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, ß-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Péptidos/genética , Péptidos/metabolismo , Espectrometría de Fluorescencia , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND/AIMS: Human Cytochrome P450 (CYP) comprises a multigene family of microsomal enzymes that metabolize a wide variety of xenobiotics, including drugs and carcinogens. Although the a number of CYP enzymes were also detected in epithelial cells along the gastrointestinal tract, little is known about the expression of CYP genes in gastric tissue. METHODOLOGY: In this study, the expression patterns of CYP isoforms was investigated in a total of 14 antral biopsy tissues obtained from the patients with either chronic gastritis (n = 6) or cancer (n = 8) by gene-specific real-time reverse transcriptase -PCR analyses. We employed primer sets specific for CYPs -1A1, -1A2, -2A6, -2B6, -2C, -2D6, -2E1, and -3A5. RESULTS: Among the isoforms CYP1A1, CYP2C and CYP2D6 gave rise to detectable mRNAs in all 14 gastric tissues while the mRNAs for the other CYPs were detected in some of the tissues. The expression patterns were compared to clinical parameters. There were no significant differences in the parameters between the two groups; however the mRNA expression of CYP2A6 was significantly higher in women than man (p < 0.05). CONCLUSIONS: Our data suggests that the CYP isoforms were independently expressed with respect to the pathological status in human gastric tissue.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Gastritis/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Biopsia , Distribución de Chi-Cuadrado , Cartilla de ADN , Electroforesis en Gel de Agar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A number of studies reported Mannich bases to manifest antimicrobial, cytotoxic, anticancer, anti-inflammatory, and anticonvulsant activities. A considerable number of therapeutically important cytotoxic compounds are active on DNA topoisomerases that regulate the DNA topology. In the present study we evaluated the biological activity of mono-Mannich bases, 1-aryl-3-phenethylamino-1-propanone hydrochlorides (1a-10a), and semicyclic mono-Mannich bases, 3-aroyl-4-aryl-1-phenethyl-4-piperidinols (1b-9b), synthesized in our laboratory. We employed androgen-independent human prostate cancer cells (PC-3) to assess the cytotoxicity of the compounds and extended the biological activity evaluation to cover supercoil relaxation assays of mammalian type I topoisomerases. Our results showed that the compounds had cytotoxicity within the 8.2-32.1 microM range, while two compounds gave rise to a comparable average value in topo I interference of 42% and 40% for 10a (with a hydroxy substituent on the phenyl ring from mono-Mannich bases) and 5b (with a fluoro substituent on the phenyl ring from the semicyclic mono-Mannich base series, piperidinols), respectively.
Asunto(s)
ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Piperidinas/farmacología , Propano/análogos & derivados , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo I/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Piperidinas/química , Propano/química , Propano/farmacología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway. METHODS: Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells. RESULTS AND CONCLUSIONS: Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Elipticinas/farmacología , Telomerasa/genética , Homeostasis del Telómero/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/química , Línea Celular , Elipticinas/química , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación Fluorescente in SituRESUMEN
Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H(2) receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Inhibidores de Topoisomerasa I , Animales , Antineoplásicos/química , Bencimidazoles/química , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Células HeLa , Humanos , Análisis Espectral , Relación Estructura-Actividad , Neoplasias del Cuello Uterino/patologíaRESUMEN
Chalcones (1,3-diaryl-2-propen-1-ones) are alpha, beta-unsaturated ketones with cytotoxic and anticancer properties. Several reports have shown that compounds with cytotoxic properties may also interfere with DNA topoisomerase functions. Five derivatives of 4'-hydroxychalcones were examined for cytotoxicity against transformed human T (Jurkat) cells as well as plasmid supercoil relaxation experiments using mammalian DNA topoisomerase I. The compounds were 3-phenyl-1-(4'-hydroxyphenyl)-2-propen-1-one (I), 3-(p-methylphenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (II), 3-(p-methoxyphenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (III), 3-(p-chlorophenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (IV), and 3-(2- thienyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (V). The order of the cytotoxicity of the compounds was; IV > III > II > I > V. Compound IV, had the highest Hammett and log P values (0.23 and 4.21, respectively) and exerted both highest cytotoxicity and strongest DNA topoisomerase I inhibition. Compounds I and II gave moderate interference with the DNA topoisomerase I while III & V did not interfere with the enzyme.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Chalconas/síntesis química , Chalconas/farmacología , Inhibidores de Topoisomerasa I , Animales , Antineoplásicos/química , Bovinos , Línea Celular , Chalconas/química , ADN-Topoisomerasas de Tipo I/metabolismo , Humanos , Concentración 50 Inhibidora , Células Jurkat , Plásmidos/metabolismoRESUMEN
DNA topoisomerases are important targets for cancer chemotherapy. We investigated the effects of a methanolic extract of Helichrysum pamphylicum on mammalian DNA topoisomerase I via in vitro plasmid supercoil relaxation assays. The extracts manifested a considerable inhibition of the enzyme's activity in a dose-dependent manner. We also performed a HPLC analysis to identify the flavonoid content of the H. pamphylicum extract and tested the identified flavonoids; luteolin, luteolin-4-glucoside, naringenin, helichrysinA and isoquercitrin, on DNA topoisomerase I activity. The measurement of the total antioxidant capacity of the flavonoid standards suggested that the topoisomerase inhibition might be correlated with the antioxidant capacity of the plant.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Helichrysum/química , Inhibidores de Topoisomerasa I , Animales , Cromatografía Liquida , ADN Superhelicoidal/efectos de los fármacos , Inhibidores Enzimáticos/química , Flavonoides/química , Cinética , Mamíferos , Extractos Vegetales/farmacología , Plásmidos/efectos de los fármacosRESUMEN
Cytochrome P450 (CYP) is a heme-containing enzyme superfamily metabolizing a wide variety of xenobiotics, including drugs and carcinogens. The majority of CYP genes are expressed in the liver, however, some CYP isoforms are also reported for a number of extra hepatic tissues. We analyzed Cytochrome P450-2A6, -3A5 and -4B1 mRNAs using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) in a total of 21 homogenized prostate tissues with or without malignancy. We detected a consistent expression of CYP2A6 and CYP3A5 in all, and of CYP4B1 in some (11/21) of the samples at mRNA level. Neither the histopathological status nor the smoking habit of the individuals affected CYP4B1 expression. Our results reflect possible roles for these particular CYPs in therapy and protection of prostate tissue.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP3A/genética , Próstata/enzimología , ARN Mensajero/genética , Biopsia , Citocromo P-450 CYP2A6 , Cartilla de ADN , ADN Complementario/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Masculino , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Benzimidazole is one of the most important heterocyclic groups manifesting various biological properties, such as antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Several benzimidazole derivatives are also active as inhibitors of type I DNA topoisomerases. In this study, three 1H-benzimidazole derivatives with different electronic characteristics at position 5-, namely 5-chloro-4-(1H-benzimidazole-2-yl)phenol (Cpd I), 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) and 4-(1H-benzimidazole-2-yl)phenol (Cpd III), were synthesized and evaluated for their effects on mammalian type I DNA topoisomerase activity using quantitative in vitro plasmid supercoil relaxation assays. For the structure elucidation of the compounds, melting points, UV, IR, 1H NMR, 13C NMR, mass spectral data and elemental analyses were interpreted. Among the compounds, 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) manifested relatively potent topoisomerase I inhibition.
Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Inhibidores de Topoisomerasa I , Animales , ADN-Topoisomerasas de Tipo I/metabolismo , Electroforesis en Gel de Agar , Espectroscopía de Resonancia Magnética , Estructura Molecular , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA- and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Acetiltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN , Humanos , Unión Proteica , Factores de Transcripción/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismoRESUMEN
A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks.
Asunto(s)
Roturas del ADN , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo I/química , Gosipol/química , Animales , Activación Enzimática , Pruebas de Enzimas , Estabilidad de Enzimas , Sustancias Macromoleculares/química , Plásmidos/química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/químicaRESUMEN
Telomeres are specialized DNA-protein complexes found at the tips of linear chromosomes. In this study, we investigated the effects of oxidative stress on telomeric length distribution of proliferating vascular smooth muscle cells following balloon injury in single or combined treatment of rabbits with either buthionine sulfoximine or taurine. Exposure to oxidative stress increased the balloon injury whereas taurine treatment significantly diminished L-buthionine-sulfoximine-related intimal hyperplasia. Our results also showed that both variables had a significant influence on mean telomeric length distribution.
Asunto(s)
Angioplastia de Balón/efectos adversos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Estrés Oxidativo , Telómero/patología , Lesiones del Sistema Vascular/etiología , Animales , Arterias/anatomía & histología , Arterias/efectos de los fármacos , Arterias/lesiones , Aterosclerosis/patología , Butionina Sulfoximina/farmacología , Proliferación Celular/efectos de los fármacos , Glutatión/sangre , Glutatión/efectos de los fármacos , Disulfuro de Glutatión/sangre , Disulfuro de Glutatión/efectos de los fármacos , Glutatión Peroxidasa/sangre , Glutatión Peroxidasa/efectos de los fármacos , Hiperplasia/patología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/lesiones , Miocitos del Músculo Liso/patología , Conejos , Taurina/farmacología , Telómero/efectos de los fármacos , Túnica Íntima/efectos de los fármacos , Túnica Íntima/patología , Lesiones del Sistema Vascular/prevención & controlRESUMEN
We investigated the effect of extremely low-frequency electromagnetic field (ELF-EMF) with pulse trains exposure on lipid peroxidation, and, hence, oxidative stress in the rat liver tissue. The parameters that we measured were the levels of plasma alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as plasma albumin, bilirubin, and total protein levels in 30 adult male Wistar rats exposed to ELF. We also determined the percentage of apoptotic and necrotic cells of the kidney extracts from the animals by flow cytometry method. Apoptotic cell death was further characterized by monitoring DNA degradation using gel electrophoresis. The results showed an increase in the levels of oxidative stress indicators, and the flow cytometric data suggested a possible relationship between the exposure to magnetic field and the cell death. We showed significantly lower necrotic cell percentages in experimental animals compared to either unexposed or sham control groups. However, DNA ladder analyses did not differentiate between the groups. Our results were discussed in relation to the response of biological systems to EMF.