RESUMEN
BACKGROUND: The present study sought to evaluate the efficacy of N-acetylcysteine amide (NACA) eye drops in reversing the cataract formation induced by sodium selenite in male Wistar rat pups. METHODS: Forty male Wistar rat pups were randomly divided into a control group, an N-acetylcysteine amide-only group, a sodium selenite-induced cataract group, and a NACA-treated sodium selenite-induced cataract group. Sodium selenite was injected intraperitoneally on postpartum day 10, whereas N-acetylcysteine amide was injected intraperitoneally on postpartum days 9, 11, and 13 in the respective groups. Cataracts were evaluated at the end of week 2 (postpartum day 14) when the rat pups opened their eyes. N-acetylcysteine amide eye drops were administered beginning on week 3 until the end of week 4 (postpartum days 15 to 30), and the rats were sacrificed at the end of week 4. Lenses were isolated and examined for oxidative stress parameters such as glutathione, lipid peroxidation, and calcium levels along with the glutathione reductase and thioltransferase enzyme activities. Casein zymography and Western blot of m-calpain were performed using the water soluble fraction of lens proteins. RESULTS: Morphological examination of the lenses in the NACA-treated group indicated that NACA was able to reverse the cataract grade. In addition, glutathione level, thioltransferase activity, m-calpain activity, and m-calpain level (as assessed by Western blot) were all significantly higher in the NACA-treated group than in the sodium selenite-induced cataract group. Furthermore, sodium selenite- injected rat pups had significantly higher levels of malondialdehyde, glutathione reductase enzyme activity, and calcium levels, which were reduced to control levels upon treatment with NACA. CONCLUSIONS: The data suggest that NACA has the potential to significantly improve vision and decrease the burden of cataract-related loss of function. Prevention and reversal of cataract formation could have a global impact. Development of pharmacological agents like NACA may eventually prevent cataract formation in high-risk populations and may prevent progression of early-stage cataracts. This brings a paradigm shift from expensive surgical treatment of cataracts to relatively inexpensive prevention of vision loss.
Asunto(s)
Acetilcisteína/análogos & derivados , Catarata/prevención & control , Cristalino/metabolismo , Estrés Oxidativo , Acetilcisteína/administración & dosificación , Animales , Western Blotting , Catarata/inducido químicamente , Catarata/diagnóstico , Modelos Animales de Enfermedad , Cristalino/efectos de los fármacos , Masculino , Soluciones Oftálmicas , Ratas , Ratas Wistar , Ácido Selenioso/toxicidadRESUMEN
BACKGROUND: Sutherlandia frutescens (L.) R.Br. (SF) is a South African plant that is widely used to treat stress, infections, cancer, and chronic diseases, many of which involve oxidative stress. The aim of the study was to quantitatively assess the antioxidant potential of SF extracts in cell-free system as well as in cell lines. METHODS: Dried SF vegetative parts were extracted using six different solvents, and the extracts were assessed for total phenolic and flavonoid contents, total reducing power, iron chelating capacity, and free radical scavenging power, including, scavenging of hydroxyl radicals, superoxide anions, nitric oxide, and hydrogen peroxide. We further investigated the freeze-dried hot water extract of SF (SFE) to assess its effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), an organic peroxide. Three different cell lines: Chinese hamster ovary (CHO), human hepatoma (HepaRG), and human pulmonary alveolar carcinoma (A549) cells, were employed to determine cell viability, intracellular reactive oxygen species (ROS) levels, and reduced to oxidized glutathione levels (GSH/GSSG). RESULTS: The results indicated that: (1) SF extracts have significant antioxidant potential that is dependent upon the nature of the extraction solvent and (2) SFE protects against tBHP-induced oxidative stress in cells by scavenging ROS and preserving intracellular GSH/GSSG. CONCLUSION: Oxidative stress is implicated in a number of disorders, and due to the public's concerns about synthetic antioxidants, various natural antioxidants are being explored for their therapeutic potential. Our findings support claims for S. frutescens being a promising adjunctive therapeutic for oxidative stress-related health problems.
Asunto(s)
Antioxidantes/farmacología , Células/efectos de los fármacos , Fabaceae/química , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Animales , Antioxidantes/química , Células CHO , Línea Celular , Supervivencia Celular , Células/metabolismo , Cricetulus , Glutatión/metabolismo , Humanos , Oxidación-Reducción , Extractos Vegetales/química , Sustancias Protectoras/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Liposomes have been widely used as a drug delivery vehicle, and currently, more than 10 liposomal formulations are approved by the Food and Drug Administration for clinical use. However, upon targeting, the release of the liposome-encapsulated contents is usually slow. We have recently demonstrated that contents from appropriately formulated liposomes can be rapidly released by the cancer-associated enzyme matrix metalloproteinase-9 (MMP-9). Herein, we report our detailed studies to optimize the liposomal formulations. By properly selecting the lipopeptide, the major lipid component, and their relative amounts, we demonstrate that the contents are rapidly released in the presence of cancer-associated levels of recombinant human MMP-9. We observed that the degree of lipid mismatch between the lipopepides and the major lipid component profoundly affects the release profiles from the liposomes. By utilizing the optimized liposomal formulations, we also demonstrate that cancer cells (HT-29) which secrete low levels of MMP-9 failed to release a significant amount of the liposomal contents. Metastatic cancer cells (MCF7) secreting high levels of the enzyme rapidly release the encapsulated contents from the liposomes.
Asunto(s)
Liposomas/química , Liposomas/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Adenocarcinoma/enzimología , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Femenino , Fluoresceínas/análisis , Humanos , Lipopéptidos/síntesis química , Lipopéptidos/químicaRESUMEN
Matrix metalloproteinases (MMPs) constitute a class of extracellular-matrix-degrading enzymes overexpressed in many cancers and contribute to the metastatic ability of the cancer cells. We have recently demonstrated that liposomal contents can be released when triggered by the enzyme MMP-9. Herein, we report the results of our mechanistic studies of the MMP-9-triggered release of liposomal contents. We synthesized peptides containing the cleavage site for MMP-9 and conjugated them with fatty acids to prepare the corresponding lipopeptides. By employing circular dichroism (CD) spectroscopy, we demonstrated that the lipopeptides, when incorporated into liposomes, are demixed in the lipid bilayers and generate triple-helical structures. MMP-9 cleaves the triple-helical peptides, leading to the release of the liposomal contents. Other MMPs, which cannot hydrolyze triple-helical peptides, fail to release the contents from the liposomes. We also observed that the rate and extent of release of the liposomal contents depend on the mismatch between the acyl chains of the synthesized lipopeptide and phospholipid components of the liposomes. CD spectroscopic studies imply that the observed differences in the release reflect the ability of the liposomal membrane to anneal the defects following the enzymatic cleavage of the liposome-incorporated lipopeptides.
Asunto(s)
Lipoproteínas/química , Liposomas/química , Metaloproteinasa 9 de la Matriz/química , Dicroismo Circular , Fluoresceínas/química , Humanos , Hidrólisis , Lipoproteínas/síntesis química , Péptidos/síntesis química , Péptidos/química , Estructura Secundaria de ProteínaRESUMEN
A series of bis-(arylsulfonamide) hydroxamate inhibitors were synthesized. These compounds exhibit good potency against MMP-7 and MMP-9 depending on the nature, steric bulk, and substitution pattern of the substituents in the benzene ring. In general, the preliminary structure-activity relationships (SAR) suggest that among the DAPA hydroxamates (i) electron-rich benzene rings of the sulfonamides may produce better inhibitors than electron-poor analogs. However, potential H-bond acceptors can reverse the trend depending on the isozyme; (ii) isozyme selectivity between MMP-7 and -9 can be conferred through steric bulk and substitution pattern of the substituents in the benzene ring, and (iii) the MMP-10 inhibition pattern of the compounds paralleled that for MMP-9.
Asunto(s)
Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Diseño de Fármacos , Ácidos Hidroxámicos/química , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over the counter antipyretic and analgesic medications. It is safe at therapeutic doses, but its overdose can result in severe hepatotoxicity, a leading cause of drug-induced acute liver failure in the USA. Depletion of glutathione (GSH) is one of the initiating steps in APAP-induced hepatotoxicity; therefore, one strategy for restricting organ damage is to restore GSH levels by using GSH prodrugs. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an acetaminophen overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and I.V. administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteine amide (NACA), a novel antioxidant with higher bioavailability, and compared it with NAC in APAP-induced hepatotoxicity in C57BL/6 mice. Our results showed that NACA is better than NAC at a low dose (106mg/kg) in preventing oxidative stress and protecting against APAP-induced damage. NACA significantly increased GSH levels and the GSH/GSSG ratio in the liver to 66.5% and 60.5% of the control, respectively; and it reduced the level of ALT by 30%. However, at the dose used, NAC was not effective in combating the oxidative stress induced by APAP. Thus, NACA appears to be better than NAC in reducing the oxidative stress induced by APAP. It would be of great value in the health care field to develop drugs like NACA as more effective and safer options for the prevention and therapeutic intervention in APAP-induced toxicity.
Asunto(s)
Acetaminofén/antagonistas & inhibidores , Acetaminofén/toxicidad , Acetilcisteína/análogos & derivados , Analgésicos no Narcóticos/toxicidad , Antídotos/farmacología , Acetilcisteína/farmacología , Alanina Transaminasa/metabolismo , Animales , Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Glutamato Deshidrogenasa/metabolismo , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
A novel strategy of blocking the active site accessibility of MMP-9 by "multi-prong" surface binding groups is described.
Asunto(s)
Cobre/química , Inhibidores Enzimáticos/farmacología , Iminoácidos/química , Inhibidores de la Metaloproteinasa de la Matriz , Compuestos Organometálicos/farmacología , Inhibidores Enzimáticos/química , Metaloproteinasa 10 de la Matriz , Metaloproteinasa 9 de la Matriz/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/química , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Peptide analogues of Tendamistat which include the most essential residues linked by novel omega-amino acids (X,Y,Z: H2N-(CH2)n-CO(2)H, where n=2-10) were designed, synthesized (Ac-Tyr(15)-X-Trp(18)-Arg(19)-Tyr(20)-Y-Thr(55)-Z-Asp(58)-Gly(59)-Tyr(60)-Ile(61)-Gly(62)-NH2), and analyzed for alpha-amylase inhibitory activity. Native dipeptide spacers sometimes were left intact at X and Z. Analogues demonstrated competitive inhibition with K(i) values ranging from 23 to 767 microM. 8-Aminooctanoic acid was the optimal linker at Y, while longer linkers were favored at the other positions.
Asunto(s)
Aminoácidos/química , Inhibidores Enzimáticos/química , Péptidos , Péptidos/química , alfa-Amilasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Humanos , Estructura Molecular , Péptidos/síntesis química , Péptidos/metabolismo , Unión Proteica , alfa-Amilasas/metabolismoRESUMEN
Manganese (Mn) is an essential trace element required for normal cellular functioning. However, overexposure of Mn can be neurotoxic resulting in the development of manganism, a syndrome that resembles Parkinson׳s disease. Although the pathogenetic basis of this disorder is unclear, several studies indicate that it is mainly associated with oxidative stress and mitochondrial energy failure. Therefore, this study is focused on (1) investigating the oxidative effects of Mn on neuroblastoma cells (SHSY5Y) and (2) elucidating whether a novel thiol antioxidant, N-acetylcysteineamide (NACA), provides any protection against Mn-induced neurotoxicity. Reactive oxygen species (ROS) were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and activities of glutathione reductase (GR) and glutathione peroxidase (GPx) were altered in the Mn-treated groups. Loss of mitochondrial membrane potential, as assessed by flow cytometry and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. However, pretreatment with NACA protected against Mn-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and mitochondrial membrane potential. NACA can potentially be developed into a promising therapeutic option for Mn-induced neurotoxicity. This article is part of a Special Issue entitled SI: Metals in neurodegeneration.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Manganeso/toxicidad , Estrés Oxidativo/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Neuroblastoma/patología , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. Despite being safe at therapeutic doses, an accidental or intentional overdose can result in severe hepatotoxicity; a leading cause of drug-induced liver failure in the U.S. Depletion of glutathione (GSH) is implicated as an initiating event in APAP-induced toxicity. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an APAP overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and intravenous administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteineamide (NACA), a novel antioxidant, with higher bioavailability and compared it with NAC in APAP-induced hepatotoxicity in a human-relevant in vitro system, HepaRG. Our results indicated that exposure of HepaRG cells to APAP resulted in GSH depletion, reactive oxygen species (ROS) formation, increased lipid peroxidation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase release. Both NAC and NACA protected against APAP-induced hepatotoxicity by restoring GSH levels, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. However, NACA was better than NAC at combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA in protecting cells against APAP-induced toxicity suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose.
Asunto(s)
Acetaminofén/efectos adversos , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Analgésicos no Narcóticos/efectos adversos , Antídotos/farmacología , Carcinoma Hepatocelular/metabolismo , Sobredosis de Droga/tratamiento farmacológico , Depuradores de Radicales Libres/farmacología , Neoplasias Hepáticas/metabolismo , Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Sobredosis de Droga/metabolismo , Sobredosis de Droga/patología , Humanos , Peroxidación de Lípido/efectos de los fármacos , Neoplasias Hepáticas/patología , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Diesel exhaust particles (DEPs), a by-product of diesel engine exhaust (DEE), are known to produce pro-oxidative and pro-inflammatory effects, thereby leading to oxidative stress-induced damage. Given the key role of DEPs in inducing oxidative stress, we investigated the role of DEPs in disrupting the integrity and function of immortalized human brain microvascular endothelial cells (HBMVEC). To study this, HBMVEC cells were exposed to media containing three different concentrations of DEPs or plain media for 24h. Those exposed to DEPs showed significantly higher oxidative stress than the untreated group, as indicated by the glutathione (GSH) and malondialdehyde (MDA) levels, and the glutathione peroxidase and glutathione reductase activities. DEPs also induced oxidative stress-related disruption of the HBMVEC cells monolayer, as measured by trans-epithelial electrical resistance. Taken together, these data suggest that DEPs induce cell death and disrupt the function and integrity of HBMVEC cells, indicating a potential role of DEPs in neurotoxicities.
Asunto(s)
Encéfalo/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Microvasos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Encéfalo/irrigación sanguínea , Línea Celular Transformada , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dextranos/metabolismo , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Humanos , Malondialdehído/metabolismo , Microvasos/metabolismo , Microvasos/patologíaRESUMEN
We offer a novel methodology for formulating liposomes by incorporating sequence-specific collagen-mimetic peptides such that they are specifically "uncorked" by a matrix metalloproteinase, MMP-9. By encapsulating carboxyfluorescein (as a self-quenching fluorescent dye), we demonstrate that the time-dependent release of the dye from liposomes is due to the specific enzymatic cleavage of the surface-exposed collagen-mimetic peptides. The specificity of such cleavage is attested by the fact that the liposomal "uncorking" and their content release occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the trypsin cleavage site. The mechanistic details underlying the formulations of liposomes and their enzyme-selective "uncorking" and content release are discussed. Arguments are presented that such liposomes can be fine-tuned to serve as the drug delivery vehicles for the detection and treatment of various human diseases, which occur due to the overexpression of a variety of pathogenic matrix metalloproteinases.
Asunto(s)
Liposomas/química , Liposomas/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Secuencia de Aminoácidos , Biomimética , Colágeno/química , Colágeno/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/metabolismo , Péptidos/química , Péptidos/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Temperatura de Transición , Tripsina/metabolismoRESUMEN
Sulfonamide derivatives serve as potent inhibitors of carbonic anhydrases (CAs), and a few such inhibitors have been currently used as drugs for the treatment of different pathogenic conditions in humans. In pursuit of designing the isozyme-specific inhibitors of human CAs, we observed that the fluorescence spectral properties and binding profiles of a fluorogenic sulfonamide derivative, 5-(dimethylamino)-1-naphthalenesulfonamide (dansylamide, DNSA), were markedly different between the recombinant forms of human carbonic anhydrase I (hCA I) and II (hCA II). The kinetic evaluation of the overall microscopic pathways for the binding of DNSA to hCA I versus hCA II revealed that the protein isomerization step served as a major determinant of the above discrepancy. Arguments are presented that the detailed structural-functional investigations of enzyme-ligand interactions may provide insights into designing the isozyme-specific inhibitors of CAs.