RESUMEN
Purpose: Central serous chorioretinopathy (CSCR) has been associated with oxidative stress-related risk factors. The objective of this study was to optimize an analytical method for evaluating the oxidative stress biomarker malondialdehyde (MDA) in human tears and determine its level in the tears of patients with CSCR. Methods: In this pilot study, tear samples were obtained from 34 healthy donors and 31 treatment-naïve CSCR male patients (eight with acute CSCR and 23 with chronic CSCR). Two analytical methods based on high-performance liquid chromatography followed by fluorescence detection were evaluated, with either 2-thiobarbituric derivative (TBA) or 2-aminoacridone (2-AA). Activity of CSCR was defined by the serous retinal detachment (SRD) height, which was measured by two independent observers on spectral-domain optical coherence tomography. Results: The 2-AA method showed higher sensitivity and precision compared to the TBA method. When the 2-AA method was applied to tears from healthy donors, the levels of MDA were statistically significantly higher in men compared to women (mean ± standard deviation, SD: 9,914 nM ± 6,126 versus 4,635 nM ± 1,173, p = 0.006). No difference was found in tear MDA levels between male patients with CSCR and age-matched control men (p = 0.17). However, MDA levels were statistically significantly higher in acute compared to chronic CSCR cases (mean ± SD: 12,295 nM ± 8,495 versus 6,790 ± 3,969 nM, p = 0.03). Additionally, there was a correlation between MDA levels and RPE leakage, quantified by the height of the serous retinal detachment (p = 0.02, r = 0.40). Conclusions: Levels of MDA in tears, measured with an optimized analytical method, correlate with RPE leakage in CSCR.
Asunto(s)
Coriorretinopatía Serosa Central/metabolismo , Coriorretinopatía Serosa Central/patología , Malondialdehído/metabolismo , Estrés Oxidativo , Lágrimas/metabolismo , Adulto , Aminoacridinas/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Coriorretinopatía Serosa Central/complicaciones , Coriorretinopatía Serosa Central/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Desprendimiento de Retina/complicaciones , Desprendimiento de Retina/diagnóstico por imagen , Tiobarbitúricos/metabolismo , Tomografía de Coherencia ÓpticaRESUMEN
The metabolism of antitumor-active 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) has been investigated widely over the last decade but some aspects of molecular mechanisms of its metabolic transformation are still not explained. In the current work, we have reported a direct and rapid analytical tool for better prediction of C-1311 metabolism which is based on electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS). Simulation of the oxidative phase I metabolism of the compound was achieved in a simple electrochemical thin-layer cell consisting of three electrodes (ROXY™, Antec Leyden, the Netherlands). We demonstrated that the formation of the products of N-dealkylation reactions can be easily simulated using purely instrumental approach. Newly reported products of oxidative transformations like hydroxylated or oxygenated derivatives become accessible. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass ion data and tandem mass spectrometry experiments. In silico prediction of main sites of C-1311 metabolism was performed using MetaSite software. The compound was evaluated for cytochrome P450 1A2-, 3A4-, and 2D6-mediated reactions. The results obtained by EC were also compared and correlated with those of reported earlier for conventional in vitro enzymatic studies in the presence of liver microsomes and in the model peroxidase system. The in vitro experimental approach and the in silico metabolism findings showed a quite good agreement with the data from EC/ESI-MS analysis. Thus, we conclude here that the electrochemical technique provides the promising platform for the simple evaluation of drug metabolism and the reaction mechanism studies, giving first clues to the metabolic transformation of pharmaceuticals in the human body.
Asunto(s)
Aminoacridinas/metabolismo , Antineoplásicos/metabolismo , Fenómenos Bioquímicos/fisiología , Simulación por Computador , Sistema Enzimático del Citocromo P-450/metabolismo , Técnicas Electroquímicas/métodos , Electroquímica/métodos , Electrodos , Humanos , Inactivación Metabólica/fisiología , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445-450 nm for the substrate, λem 550 nm for the product). Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino)-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our ß-alanyl aminopeptidase substrate, 2-(N- ß-alanylamino)-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.
Asunto(s)
Aminoacridinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Serratia marcescens/metabolismo , beta-Alanina/metabolismo , Espectrometría de FluorescenciaRESUMEN
Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in â¼1-2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1-2 h.
Asunto(s)
Aminoacridinas/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Disacáridos/análisis , Glicosaminoglicanos/metabolismo , Espectrometría de Masas/métodos , Disacáridos/metabolismo , Fluorescencia , Coloración y EtiquetadoRESUMEN
AIM: To examine whether CYP3A4 overexpression influences the metabolism of anticancer agent imidazoacridinone C-1311 in CHO cells and the responses of the cells to C-1311. METHODS: Wild type CHO cells (CHO-WT), CHO cells overexpressing cytochrome P450 reductase (CPR) [CHO-HR] and CHO cells coexpressing CPR and CYP3A4 (CHO-HR-3A4) were used. Metabolic transformation of C-1311 and CYP3A4 activity were measured using RP-HPLC. Flow cytometry analyses were used to examine cell cycle, caspase-3 activity and cell apoptosis. The expression of pH 6.0-dependent ß-galactosidase (SA-ß-gal) was studied to evaluate accelerated senescence. ROS generation was analyzed with CM-H2 DCFDA staining. RESULTS: CYP3A4 overexpression did not change the metabolism of C-1311 in CHO cells: the levels of all metabolites of C-1311 increased with the exposure time to a similar extent, and the differences in the peak level of the main metabolite M3 were statistically insignificant among the three CHO cell lines. In CHO-HR-3A4 cells, C-1311 effectively inhibited CYP3A4 activity without affecting CYP3A4 protein level. In the presence of C-1311, CHO-WT cells underwent rather stable G2/M arrest, while the two types of transfected cells only transiently accumulated at this phase. C-1311-induced apoptosis and necrosis in the two types of transfected cells occurred with a significantly faster speed and to a greater extent than in CHO-WT cells. Additionally, C-1311 induced ROS generation in the two types of transfected cells, but not in CHO-WT cells. Moreover, CHO-HR-3A4 cells that did not die underwent accelerated senescence. CONCLUSION: CYP3A4 overexpression in CHO cells enhances apoptosis induced by C-1311, whereas the metabolism of C-1311 is minimal and does not depend on CYP3A4 expression.
Asunto(s)
Aminoacridinas/metabolismo , Antineoplásicos/metabolismo , Apoptosis/fisiología , Citocromo P-450 CYP3A/biosíntesis , Regulación Enzimológica de la Expresión Génica , Aminoacridinas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.
Asunto(s)
Acridinas/metabolismo , Acridinas/farmacología , Aminoacridinas/metabolismo , Aminoacridinas/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Glucuronosiltransferasa/metabolismo , Triazoles/metabolismo , Triazoles/farmacología , Neoplasias del Cuello Uterino/enzimología , Biotransformación , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Glucurónidos/metabolismo , Glucurónidos/farmacología , Glucuronosiltransferasa/genética , Células HeLa , Humanos , Concentración 50 Inhibidora , Transfección , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Copper-based fungicides have been widely used against several grapevine (Vitis vinifera L.) diseases since the late 1800s when the Bordeaux mixture was developed, but their intensive use has raised phytotoxicity concerns. In this study, physiological, biochemical and molecular approaches were combined to investigate the impacts of copper in grape cells and how it is transported and compartmented intracellularly. Copper reduced the growth and viability of grape cells (CSB, Cabernet Sauvignon Berry) in a dose-dependent manner above 100 µM and was accumulated in specific metal ion sinks. The copper-sensitive probe Phen Green SK was used to characterize copper transport across the plasma membrane of CSB cells. The transport system (K(m) = 583 µM; V(max) = 177 × 10(-6) %ΔF min(-1) protoplast(-1)) was regulated by copper availability in the culture medium, stimulated by Ca(2+) and inhibited by Zn(2+). The pH-sensitive fluorescent probe ACMA (9-amino-6-chloro-2-methoxyacridine) was used to evaluate the involvement of proton-dependent copper transport across the tonoplast. Cu(2+) compartmentation in the vacuole was dependent on the transmembrane pH gradient generated by both V-H(+)-ATPase and V-H(+)-pyrophosphatase (PPase). High copper levels in the growth medium did not affect the activity of V-H(+)-PPase but decreased the magnitude of the H(+) gradient generated by V-H(+)-ATPase. Expression studies of VvCTr genes showed that VvCTr1 and VvCTr8 were distinctly affected by CuSO(4) availability in grape cell cultures and that both genes were highly expressed in the green stage of grape berries.
Asunto(s)
Cobre/metabolismo , Regulación de la Expresión Génica de las Plantas , Células Vegetales/metabolismo , Vitis/metabolismo , Aminoacridinas/metabolismo , Transporte Biológico , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular , Sulfato de Cobre/farmacología , Medios de Cultivo/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Frutas/citología , Frutas/metabolismo , Genes de Plantas , Concentración de Iones de Hidrógeno , Pirofosfatasa Inorgánica/metabolismo , Células Vegetales/efectos de los fármacos , Células Vegetales/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Bombas de Protones/metabolismo , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/efectos de los fármacos , Vacuolas/enzimología , Vacuolas/metabolismo , Vitis/efectos de los fármacos , Vitis/enzimología , Vitis/genéticaRESUMEN
5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 µl · min⻹ · mg protein⻹, for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 µl · min⻹ · mg protein⻹. In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.
Asunto(s)
Acridinas/metabolismo , Aminoacridinas/metabolismo , Antineoplásicos/metabolismo , Glucuronosiltransferasa/metabolismo , Intestinos/enzimología , Hígado/enzimología , Triazoles/metabolismo , Biotransformación , Catálisis , Cromatografía Líquida de Alta Presión , Glucurónidos/metabolismo , Glucuronosiltransferasa/genética , Humanos , Cinética , Microsomas Hepáticos/enzimología , Modelos Biológicos , Mutación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing lysosomal photodestruction of normal breast epithelial cells. Thus, MDR modalities including ABCG2-dependent drug sequestration within EVs can be rationally converted to a pharmacologically lethal Trojan horse to selectively eradicate MDR cancer cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/metabolismo , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Aminoacridinas/metabolismo , Aminoacridinas/farmacología , Antineoplásicos/farmacología , Transporte Biológico/efectos de los fármacos , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Macrólidos/farmacología , Proteínas de Neoplasias/genética , Fármacos Fotosensibilizantes/farmacología , Transporte de Proteínas , Topotecan/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The narrow "therapeutic window" of anti-tumour therapy may be the result of drug metabolism leading to the activation or detoxification of antitumour agents. The aim of this work is to examine (i) whether the diminished toxicity of a potent antitumour drug, C-1748, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine, compared with its 4-demethyl analogue, C-857, results from the differences between the metabolic pathways for the two compounds and (ii) the impact of reducing and/or hypoxic conditions on studied metabolism. We investigated the metabolites of C-1748 and C-857 formed in rat and human liver microsomes, with human P450 reductase (POR) and in HepG2 cells under normoxia and hypoxia. The elimination rate of C-1748 from POR knockout mice (HRN) was also evaluated. Three products, 1-amino-9-hydroxyethylaminoacridine, 1-aminoacridinone and a compound with an additional 6-membered ring, were identified for C-1748 and C-857 in all studied metabolic systems. The new metabolite was found in HepG2 cells. We showed that metabolic rate and the reactivity of metabolites of C-1748 were considerably lower than those of C-857, in all investigated metabolic models. Compared with metabolism under normoxia, cellular metabolism under hypoxia led to higher levels of 1-aminoacridine and aza-acridine derivatives of both compounds and of the 6-membered ring metabolite of C-1748. In conclusion, the crucial role of hypoxic conditions and the direct involvement of POR in the metabolism of both compounds were demonstrated. Compared with C-857, the low reactivity of C-1748 and the stability of its metabolites are postulated to contribute significantly to the diminished toxicity of this compound observed in animals.
Asunto(s)
Aminoacridinas/metabolismo , Aminoacridinas/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Nitracrina/análogos & derivados , Aminoacridinas/química , Animales , Antineoplásicos/química , Biotransformación , Técnicas de Cultivo de Célula , Hipoxia de la Célula/fisiología , Cromatografía Líquida de Alta Presión , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Noqueados , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Estructura Molecular , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/fisiología , Nitracrina/química , Nitracrina/metabolismo , Nitracrina/farmacología , Ratas , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.
Asunto(s)
Acridinas/metabolismo , Antineoplásicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxigenasas/metabolismo , Triazoles/metabolismo , Acridinas/química , Acridinas/farmacología , Aminoacridinas/química , Aminoacridinas/metabolismo , Aminoacridinas/farmacología , Animales , Antineoplásicos/farmacología , Biocatálisis/efectos de los fármacos , Biotransformación/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Isoenzimas/metabolismo , Cinética , Microsomas Hepáticos/efectos de los fármacos , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Donantes de Tejidos , Triazoles/química , Triazoles/farmacologíaRESUMEN
5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P(FMO), which is identified as an N-oxide derivative, was identical to P3(R) of liver microsomes. P3(R) was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3(R) = P(FMO). Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.
Asunto(s)
Aminoacridinas/metabolismo , Antineoplásicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Imidazoles/metabolismo , Oxigenasas/metabolismo , Aminoacridinas/química , Aminoacridinas/farmacocinética , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Biotransformación , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/química , Imidazoles/farmacocinética , Isoenzimas , Ratones , Ratones Noqueados , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Estructura Molecular , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Oxigenasas/antagonistas & inhibidores , Ratas , Especificidad por SustratoRESUMEN
Conventional spectroscopic tools such as absorption, fluorescence, and circular dichroism spectroscopy used in the study of photoinduced drug-protein interactions can yield useful information about ground-state and excited-state phenomena. However, photoinduced electron transfer (PET) may be a possible phenomenon in the drug-protein interaction, which may go unnoticed if only conventional spectroscopic observations are taken into account. Laser flash photolysis coupled with an external magnetic field can be utilized to confirm the occurrence of PET and authenticate the spin states of the radicals/radical ions formed. In the study of interaction of the model protein human serum albumin (HSA) with acridine derivatives, acridine yellow (AY) and proflavin (PF(+)), conventional spectroscopic tools along with docking study have been used to decipher the binding mechanism, and laser flash photolysis technique with an associated magnetic field (MF) has been used to explore PET. The results of fluorescence study indicate that fluorescence resonance energy transfer takes place from the protein to the acridine-based drugs. Docking study unveils the crucial role of Ser 232 residue of HSA in explaining the differential behavior of the two drugs towards the model protein. Laser flash photolysis experiments help to identify the radicals/radical ions formed in the due course of PET (PF(â¢), AY(â¢-), TrpH(â¢+), Trp(â¢)), and the application of an external MF has been used to characterize their initial spin-state. Owing to its distance dependence, MF effect gives an idea about the proximity of the radicals/radical ions during interaction in the system and also helps to elucidate the reaction mechanisms. A prominent MF effect is observed in homogeneous buffer medium owing to the pseudoconfinement of the radicals/radical ions provided by the complex structure of the protein.
Asunto(s)
Acridinas/metabolismo , Antineoplásicos/metabolismo , Campos Electromagnéticos , Albúmina Sérica/metabolismo , Acridinas/química , Algoritmos , Aminoacridinas/química , Aminoacridinas/metabolismo , Antineoplásicos/química , Sitios de Unión , Dicroismo Circular , Transporte de Electrón/efectos de la radiación , Radicales Libres/química , Radicales Libres/metabolismo , Humanos , Iones/química , Fotoquímica , Proflavina/química , Proflavina/metabolismo , Unión Proteica , Albúmina Sérica/química , Espectrometría de FluorescenciaRESUMEN
Mitochondrial membrane hyperpolarization and morphologic changes are important in inflammatory cell activation. Despite the pathophysiologic relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is available currently. The purpose of this study was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), which is a condition associated with altered mitochondrial homeostasis. We employed 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (Psi(m)) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacologic manipulation of Psi(m). We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P = 0.02) and mitochondrial mass was lower in T2DM (P = 0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, were more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P = 0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Furthermore, potentially clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Linfocitos/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/ultraestructura , Monocitos/metabolismo , Adulto , Anciano , Aminoacridinas/metabolismo , Bencimidazoles/metabolismo , Biomarcadores , Carbocianinas/metabolismo , Cardiolipinas/metabolismo , Estudios de Casos y Controles , Estudios Transversales , Femenino , Colorantes Fluorescentes/metabolismo , Humanos , Linfocitos/citología , Linfocitos/ultraestructura , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Monocitos/citología , Monocitos/ultraestructura , Reproducibilidad de los Resultados , Superóxidos/metabolismoRESUMEN
'Sono-functional' molecule 1 was prepared and its binding properties to DNA under ultrasonic irradiation were studied by UV spectra. As a result, it was shown that the binding of 1 to DNA was enhanced by ultrasound. Being compared with its precursor 2, it is clear that terminal thiol groups of 1 play an important role in specific binding to DNA.
Asunto(s)
Aminoacridinas/química , Aminoacridinas/metabolismo , ADN/metabolismo , Sonicación , Aminoacridinas/síntesis química , Animales , Bovinos , Albúmina Sérica Bovina/metabolismo , Espectrofotometría Ultravioleta , Agua/químicaRESUMEN
Heparitinase I, a key lyase enzyme essential for structural analysis of heparan sulfate (HS), degrades HS domains that are undersulfated at glucuronyl residues through an elimination mechanism. Earlier studies employed viscosimetric measurements and electrophoresis to deduce the mechanism of action of heparitinase I and two other related lyases, heparitinase II and heparitinase III. However, these findings lack molecular evidence for the intermediates formed and could not distinguish whether the cleavage occurred from the reducing end or the nonreducing end. In the current study, 2-aminoacridone (2-AMAC)-labeled HS precursor oligosaccharides of various sizes were prepared to investigate the mechanism of heparitinase I-mediated depolymerization using sensitive and quantitative methodologies. Furthermore, fluorescent (2-AMAC) tagging of HS precursor oligosaccharides allowed us to distinguish fragments that result from cleavage of the substrates at various time intervals and sites farther away from the reducing and nonreducing ends of oligosaccharide substrates. This study provides the first direct molecular evidence for a predominantly random endolytic mechanism of cleavage of HS precursor oligosaccharides by heparitinase I. This robust strategy can be adapted to deduce the mechanism of action of other heparitinases and also to deduce structural information of complex HS oligosaccharides of biological importance.
Asunto(s)
Aminoacridinas/metabolismo , Pruebas de Enzimas/métodos , Colorantes Fluorescentes/metabolismo , Liasa de Heparina/metabolismo , Heparitina Sulfato/metabolismo , Oligosacáridos/metabolismo , Coloración y Etiquetado/métodos , Biocatálisis , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Heparitina Sulfato/química , Peso Molecular , Oligosacáridos/química , Espectrometría de Masa por Ionización de Electrospray , Factores de TiempoRESUMEN
A method is presented for the electroanalytical characterization of interactions of dsDNA with a drug, under conditions that both agents are dissolved in the phosphate buffer solution and both are electroactive. Normal pulse, square wave, differential pulse, and cyclic voltammetries were employed in the measurements of the drug and dsDNA oxidation signals at carbon electrodes. UV-Vis spectroscopy was used as a non-electrochemical method to support the electroanalytical data. An anticancer drug, C-1311 (5-diethylaminoethyl-amino-8-hydroxyimidazoacridinone), has been selected for the examination. Normal pulse voltammetry was particularly useful in showing that under the conditions employed neither dsDNA nor the drug were adsorbed at the electrode surface. Necessary conditions for the appearance of the well-defined dsDNA voltammetric signal (guanine peak) are: rigorous chemical and biological purity in the cell and appropriate purity of DNA. An analysis of the obtained results confirmed that there were two modes of interaction between C-1311 and dsDNA: by intercalation and electrostatically. In the presence of excess NaCl the electrostatic interactions deteriorate. The binding constants (K (1) and K (2), respectively) and the number (n) of nucleic base pairs (bp) and the number (m) of phosphate groups (pg) interacting with one molecule of drug have been determined. For strong interactions (intercalation) the values of the binding constant, K (1), and the binding-site size, n, equal 3.7 x 10(4) M(-1) and 2.1, respectively. For the weak electrostatic interactions the K (2) and m parameters equal 0.28 x 10(4) M(-1) and 4.7. The intercalation process is rather slow and its rate (the conditions of pseudo-first-order reaction) was estimated to equal 7 x 10(-4) s(-1). The possibility of independent determination of both interacting agents was very useful in the study.
Asunto(s)
Aminoacridinas/química , Antineoplásicos/química , ADN/química , Sustancias Intercalantes/química , Aminoacridinas/metabolismo , Antineoplásicos/metabolismo , Emparejamiento Base , Sitios de Unión , Electroquímica , Electrodos , Sustancias Intercalantes/metabolismo , Cinética , Fosfatos/química , Cloruro de Sodio/química , Espectrofotometría Ultravioleta , Electricidad EstáticaRESUMEN
Measuring cytochrome c release during apoptosis provides valuable information about the nature and extent of apoptosis. Several years ago a flow cytometric method (based on selective permeabilization of the plasma membrane with digitonin) was developed that has advantages over other techniques. These experiments describe a comprehensive evaluation of that method. Apoptosis was triggered in Jurkat cells with staurosporine and then flow cytometry was used to measure three aspects of mitochondrial damage: (1) cytochrome c release (with the digitonin assay and a commercially available kit based on the same principle), using a DNA-binding dye to define cell cycle stage; (2) loss of mitochondrial cardiolipin, assessed by a decrease in 10 N-nonyl acridine orange (NAO) binding; and (3) loss of mitochondrial membrane potential, assessed by a decrease in tetramethylrhodamineethylester (TMRE) binding. The results from these three assays were compared with an antibody-based assay for cleaved caspase 3. The digitonin assay and the commercially available kit gave comparable results, showing that staurosporine caused cytochrome c release in all phases of the cell cycle and clearly defining those cells that had lost DNA due to internucleosomal DNA fragmentation. The pattern of fluorescence demonstrated that the mitochondrial apoptotic pathway was either the sole or the predominant pathway to be activated and that cytochrome c release in an individual cell was all-or-nothing. However, comparison with the other assays showed that the cytochrome c release assay underestimated the true extent of apoptosis. This was caused by the selective loss of some digitonin-treated apoptotic cells. The flow cytometry assay for cytochrome c release provides valuable information but it underestimates the percentage of apoptotic cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Citocromos c/metabolismo , Citometría de Flujo/métodos , Mitocondrias/efectos de los fármacos , Estaurosporina/farmacología , Aminoacridinas/metabolismo , Cardiolipinas/metabolismo , Caspasa 3/metabolismo , Citocromos c/aislamiento & purificación , Humanos , Células Jurkat , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Compuestos Organometálicos/metabolismo , Estaurosporina/metabolismoRESUMEN
The A421 ABCG2 genotype is a frequent polymorphism encoding the K141 transporter, which is associated with a significant decrease in transporter expression and function when compared to the wild type (wt) C421 allele encoding the Q141 ABCG2. Here we show that during the acquisition of resistance to the novel triazoloacridone antitumor agent C-1305 in lung cancer cells harboring a heterozygous C421A genotype, a marked C421 allele-specific ABCG2 gene amplification occurred. This monoallelic C421 ABCG2 gene amplification brought about the overexpression of both C421 ABCG2 mRNA and the transporter at the plasma membrane. This resulted in the lack of cellular drug accumulation due to increased efflux of both C1305 and C-1311, a fluorescent imidazoacridone homologue of C-1305, as well as marked resistance to these antitumor agents and to established ABCG2 substrates including mitoxantrone and SN-38. Consistently, the accumulation and sensitivity to these drugs were restored upon incubation with the potent and specific ABCG2 transport inhibitors Ko143 and fumitremorgin C. Moreover, upon transfection into HEK293 cells, the wt Q141 ABCG2 allele displayed a significantly decreased accumulation of C-1311 and increased resistance to C-1305, C-1311 and mitoxantrone, when compared to the K141 ABCG2 transfectant. Hence, the current study provides the first evidence that during the exposure to anticancer drugs, an allele-specific Q141 ABCG2 gene amplification occurs that confers a drug resistance advantage when compared to the K141 ABCG2. These findings have important implications for the selection and expansion of malignant anticancer drug resistant clones during chemotherapy with ABCG2 drugs.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Acridinas/farmacología , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Resistencia a Antineoplásicos/genética , Amplificación de Genes , Proteínas de Neoplasias/genética , Triazoles/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Acridinas/metabolismo , Aminoacridinas/metabolismo , Camptotecina/análogos & derivados , Camptotecina/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica , Heterocigoto , Humanos , Indoles/farmacología , Irinotecán , Mitoxantrona/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , ARN Mensajero/metabolismo , Triazoles/metabolismoRESUMEN
A real-time fluorescence assay system using a series of 9-N-(alkylamino)acridine derivatives (methyl, ethyl, n-propyl, n-butyl, n-pentyl, and benzyl) that are N-dealkylated to 9-aminoacridine (9AA) is described. The product, 9AA, is approximately 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYP1A1, 1A2, 3A4, 3A5, 1B1, 2C9, 2C19, and 2D6 indicated that N-dealkylase activity is specific for CYP1A1 and CYP2D6. CYP2D6 N-dealkylated methyl, ethyl, n-propyl, and n-butyl substrates, whereas CYP1A1 N-dealkylated these plus the n-pentyl derivative. Activities using 5 microM 9-N-(alkylamino)acridine substrates ranged from 0.1 to 0.9 pmol 9AA/min/pmol P450. Kinetic constants for CYP1A1 N-dealkylation of the 9-N-(methylamino)acridine (MAA) and 9-N-(ethylamino)acridine (EAA) were K(m) 1.09 +/- 0.68 and 0.35 +/- 0.21 microM and the V(max) 61.9 +/- 48.5 and 113.8 +/- 8.4 pmol 9AA/min/pmol CYP1A1, respectively. Kinetic constants for CYP2D6 N-dealkylation of MAA and EAA were K(m) 7.9 +/- 5.4 and 3.2 +/- 1.6 microM, and V(max) 501 +/- 35.4 and 702.7 +/- 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies (DeltaG(bind)) were calculated for MAA with CYP1A1 and CYP2D6 to be -8.266 and -7.074 kcal/mol, respectively. The DeltaG(bind) values for EAA with CYP1A1 and CYP2D6 were -8.950 and -7.618 kcal/mol, respectively. The substrates were suitable for monitoring N-dealkylase activity in microsomal preparations (human, rat, and monkey hepatic preparations) and human hepatocellular carcinoma cell suspensions. Assays were conducted by monitoring reactions either in 96-well microtiter plates using a fluorescence plate reader or in cuvettes using a spectrofluorimeter.