RESUMEN
PURPOSE: Corneal epithelial defects from trauma or surgery heal as new epithelial cells grow centripetally from the limbus and replenish the epithelium. Corneal wound healing requires cell signalling molecules. However, a topical treatment with these components is not available. Human breast milk (HBM) offers a potential, novel treatment as it contains bioactive molecules important in epithelial cell healing. This study seeks to investigate the potential of HBM in cornea wound healing. METHODS: Balb/c mice, 8-12 weeks old, were anesthetized prior to creating a 2 mm central cornea epithelial defect. Mice were randomly assigned to a treatment group: HBM, ophthalmic ointment containing neomycin, polymyxin B, dexamethasone (RxTx), or saline and treated 4x/day for 2 days. Wound area was quantified by fluorescein and ImageJ at 0, 8, 24, and 48 h post wounding and eyes used for histology, RT-qPCR, and ELISA. RESULTS: Wounded corneas treated with HBM demonstrated increased re-epithelialization at 8 h post injury compared to saline treatments. ELISA showed significantly higher Ki67 in HBM treated eyes vs. saline control at 8 h (p = 0.0278). Additionally, immunohistology revealed more Ki67 positive cells in the HBM group compared to saline at 8 h and 24 h (p = 0.0063 8 h; p = 0.0007 24 h). For inflammatory analysis, HBM group IL-1ß levels were similar to the saline group, and higher than RxTx treated eyes (p < 0.05). Immunohistochemical staining for CD11b (macrophage marker) revealed HBM-treated eyes had significantly more positive cells vs. saline. RT-qPCR of limbal stem cell markers (LESCs) revealed upregulation of Integrin αV at 8 h with HBM vs. saline. CONCLUSIONS: HBM treatment on corneas with debridement of epithelium demonstrated improved healing, cellular proliferation, and upregulation of the LESC gene transcript, integrin αV, after wounding. Future studies could investigate LESC response to different signalling molecules in HBM to better understand the efficacy of this potential therapy.
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Aldose reductase is a member of the 1B1 subfamily of aldo-keto reductase gene superfamily. The action of aldose reductase (AR) has been implicated in the pathogenesis of a variety of disease states, most notably complications of diabetes mellitus including neuropathy, retinopathy, nephropathy, and cataracts. To explore for mechanistic roles for AR in disease pathogenesis, we established mutant strains produced using Crispr-Cas9 to inactivate the AKR1B3 gene in C57BL6 mice. Phenotyping AR-knock out (ARKO) strains confirmed previous reports of reduced accumulation of tissue sorbitol levels. Lens epithelial cells in ARKO mice showed markedly reduced epithelial-to-mesenchymal transition following lens extraction in a surgical model of cataract and posterior capsule opacification. A previously unreported phenotype of preputial sebaceous gland swelling was observed frequently in male ARKO mice homozygous for the mutant AKR1B3 allele. This condition, which was shown to be accompanied by infiltration of proinflammatory CD3+ lymphocytes, was not observed in WT mice or mice heterozygous for the mutant allele. Despite this condition, reproductive fitness of the ARKO strain was indistinguishable from WT mice housed under identical conditions. These studies establish the utility of a new strain of AKR1B3-null mice created to support mechanistic studies of cataract and diabetic eye disease.
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Opacificación Capsular , Catarata , Cristalino , Animales , Masculino , Ratones , Aldehído Reductasa/genética , Opacificación Capsular/patología , Catarata/genética , Catarata/patología , Incidencia , Inflamación/patología , Cristalino/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Glándulas SebáceasRESUMEN
Aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, has been implicated in the onset and development of the ocular complications of diabetes, including cataracts and retinopathy. Despite decades of research conducted to address possible mechanisms, questions still persist in understanding if or how AR contributes to imbalances leading to diabetic eye disease. To address these questions, we created a strain of transgenic mice engineered for the overexpression of human AR (AR-Tg). In the course of monitoring these animals for age-related retinal phenotypes, we observed signs of Müller cell gliosis characterized by strong immunostaining for glial fibrillary acidic protein. In addition, we observed increased staining for Iba1, consistent with an increase in the number of retinal microglia, a marker of retinal inflammation. Compared to age-matched nontransgenic controls, AR-Tg mice showed an age-dependent loss of Brn3a-positive retinal ganglion cells and an associated decrease in PERG amplitude. Both RGC-related phenotypes were rescued in animals treated with Sorbinil in drinking water. These results support the hypothesis that increased levels of AR may be a risk factor for structural and functional changes known to accompany retinopathy in humans.
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Purpose: To determine the effect of metformin on early Nd:YAG laser treatment for posterior capsule opacification (PCO) and to explore a molecular mechanism to explain a possible protective effect of metformin against PCO. Methods: We conducted: 1) a retrospective cohort study of patient eyes undergoing phacoemulsification at our institution; and 2) laboratory investigation of the effect of metformin on the behavior of lens epithelial cells in the context of an animal model for PCO. Population-averaged Cox proportional hazards modeling was used to estimate risk for time to Nd:YAG. For laboratory studies, expression of markers for epithelial-to-mesenchymal transition (EMT) implicated in PCO pathogenesis was measured in tissue culture and following extracapsular lens extraction in a mouse model. Results: The rate of Nd:YAG laser capsulotomy was 13.1% among the 9798 eyes. Both metformin use and diabetes were protective factors for Nd:YAG laser capsulotomy in univariate analysis. However, in multivariable analysis with nondiabetics as the reference group, only metformin use among diabetics was significantly protective of Nd:YAG (hazard ratio: 0.68, 95% CI: 0.54-0.85, P = 0.0008), while eyes of patients with diabetes without metformin use did not significantly differ (P = 0.5026). Treatment of lens epithelial cells with metformin reduced the level of the EMT markers âº-SMA and pERK induced by TGF-ß2. Similarly, metformin treatment reduced âº-SMA expression in lens epithelial cells following extracapsular lens extraction in a mouse model. Conclusions: The protective effect of metformin against early Nd:YAG may relate to its ability to downregulate EMT in residual lens epithelial cells that otherwise trend toward myofibroblast development and PCO.
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Opacificación Capsular/terapia , Terapia por Láser/métodos , Láseres de Estado Sólido/uso terapéutico , Metformina/uso terapéutico , Cápsula Posterior del Cristalino/efectos de los fármacos , Capsulotomía Posterior/métodos , Complicaciones Posoperatorias/prevención & control , Anciano , Femenino , Estudios de Seguimiento , Humanos , Hipoglucemiantes/uso terapéutico , Lentes Intraoculares , Masculino , Persona de Mediana Edad , Cápsula Posterior del Cristalino/cirugía , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
Cataracts, a clouding of the eye lens, are a leading cause of visual impairment and are responsible for one of the most commonly performed surgical procedures worldwide. Although generally safe and effective, cataract surgery can lead to a secondary lens abnormality due to transition of lens epithelial cells to a mesenchymal phenotype (EMT) and opacification of the posterior lens capsular bag. Occurring in up to 40% of cataract cases over time, posterior capsule opacification (PCO) introduces additional treatment costs and reduced quality of life for patients. Studies have shown that PCO pathogenesis is driven in part by TGF-ß, signaling through the action of the family of Smad coactivators to effect changes in gene transcription. In the present study, we evaluated the ability of Smad-7, a well characterized inhibitor of TGF-ß -mediated Smad signaling, to suppress the EMT response in lens epithelial cells associated with PCO pathogenesis. Treatment of lens epithelial cells with a cell-permeable form of Smad7 variant resulted in suppressed expression of EMT markers such as alpha smooth muscle actin and fibronectin. A single application of cell-permeable Smad7 variant in the capsular bag of a mouse cataract surgery model resulted in suppression of gene transcripts encoding alpha smooth muscle actin and fibronectin. These results point to Smad7 as a promising biotherapeutic agent for prevention or substantial reduction in the incidence of PCO following cataract surgery.
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Opacificación Capsular/prevención & control , Péptidos de Penetración Celular/uso terapéutico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Productos del Gen tat/uso terapéutico , Cristalino/efectos de los fármacos , Proteína smad7/uso terapéutico , Actinas/metabolismo , Animales , Opacificación Capsular/etiología , Opacificación Capsular/patología , Catarata/complicaciones , Catarata/patología , Células Epiteliales/efectos de los fármacos , Cristalino/patología , Ratones Transgénicos , Dominios Proteicos , Proteínas Recombinantes/uso terapéuticoRESUMEN
After cataract surgery, epithelial cells lining the anterior lens capsule can transition to one of two divergent pathways, including fibrosis which leads to posterior capsular opacification (PCO), or lens fiber cell differentiation which leads to regeneration of lens material. We previously showed that the PCO response can be suppressed with aldose reductase (AR) inhibitors. In this present study we show that AR inhibition, both genetic and pharmacologic with Sorbinil, can augment the process of lens regeneration. Extracapsular lens extraction (ECLE) was carried out in C57BL/6 (WT), AR overexpression (AR-Tg), and AR knockout (ARKO) mice, and in some cases in mice treated with the AR inhibitor sorbinil. Whole eyes were harvested approximately 8 weeks after ECLE and evaluated by histological analysis and immunostaining for the fiber cell marker γ-crystallin. All eyes examined for lens regeneration were paraffin embedded for serial sectioning to produce three-dimensional reconstructed models of lens morphology and size. We observed that AR-null mice respond to ECLE by regenerating a lens-like structure with a circular shape and array of cell nuclei reminiscent of the lens bow region typical of the native mammalian lens. Although WT and AR-Tg eyes also produced some regenerated lens material after ECLE, their structures were consistently smaller than ARKO regenerated lenses. WT mice treated with sorbinil showed higher levels of lens regeneration after ECLE compared to WT mice, as assessed by size and three-dimensional morphology. Altogether, this study adds evidence for a critical role for AR in the response of lens epithelial cells to cataract extraction and lens regeneration.
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Aldehído Reductasa/metabolismo , Cristalino/fisiología , Regeneración , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/genética , Animales , Extracción de Catarata , Inhibidores Enzimáticos/farmacología , Ojo/diagnóstico por imagen , Imagenología Tridimensional , Imidazolidinas/farmacología , Cristalino/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración/efectos de los fármacosRESUMEN
Diabetes-induced hyperglycemia plays a key pathogenic role in degenerative retinal diseases. In diabetic hyperglycemia, aldose reductase (AR) is elevated and linked to the pathogenesis of diabetic retinopathy (DR) and cataract. Retinal microglia (RMG), the resident immune cells in the retina, are thought to contribute to the proinflammatory phenotype in the diabetic eye. However, we have a limited understanding of the potential role of AR expressed in RMG as a mediator of inflammation in the diabetic retina. Glycated proteins accumulate in diabetes, including Amadori-glycated albumin (AGA) which has been shown to induce a proinflammatory phenotype in various tissues. In this study, we investigated the ability of AGA to stimulate inflammatory changes to RMG and macrophages, and whether AR plays a role in this process. In macrophages, treatment with an AR inhibitor (Sorbinil) or genetic knockdown of AR lowered AGA-induced TNF-α secretion (56% and 40%, respectively) as well as cell migration. In a mouse RMG model, AR inhibition attenuated AGA-induced TNF-α secretion and cell migration (67% and 40%, respectively). To further mimic the diabetic milieu in retina, we cultured RMG under conditions of hypoxia and observed the induction of TNF-α and VEGF protein expression. Downregulation of AR in either a pharmacological or genetic manner prevented hypoxia-induced TNF-α and VEGF expression. In our animal study, increased numbers of RMG observed in streptozotocin (STZ)-induced diabetic retina was substantially lower when diabetes was induced in AR knockout mice. Thus, in vitro and in vivo studies demonstrated that AR is involved in diabetes-induced RMG activation, providing a rationale for targeting AR as a therapeutic strategy for DR.
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Diabetes Mellitus Experimental/patología , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Animales , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Regulación hacia Abajo/efectos de los fármacos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/genética , Imidazolidinas/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Células RAW 264.7 , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Retina/citología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cataract is the most frequent cause of blindness worldwide and is treated by surgical removal of the opaque lens to restore the light path to the retina. While cataract surgery is a safe procedure, some patients develop a complication of the surgery involving opacification and wrinkling of the posterior lens capsule. This process, called posterior capsule opacification (PCO), requires a second clinical treatment that can in turn lead to additional complications. Prevention of PCO is a current unmet need in the vision care enterprise. The pathogenesis of PCO involves the transition of lens epithelial cells to a mesenchymal phenotype, designated epithelial-to-mesenchymal transition (EMT). Our previous studies showed that transgenic mice designed for overexpression of human aldose reductase developed lens defects reminiscent of PCO. In the current study, we evaluated the impact of aldose reductase (AR) on expression of expression of EMT markers in the lens. Primary lens epithelial cells from AR-transgenic mice showed downregulated expression of Foxe3 and Pax6 and increased expression of α-SMA, fibronectin and snail, a pattern of gene expression typical of cells undergoing EMT. A role for AR in these changes was further confirmed when we observed that they could be normalized by treatment of cells with Sorbinil, an AR inhibitor. Smad-dependent and Smad-independent pathways are known to contribute to EMT. Interestingly, AR overexpression induced ERK but not Smad-2 activation. These results suggest that elevation of AR may lead to activation of ERK signaling and thus play a role in TGF-ß/Smad independent induction of EMT in lens epithelial cells.
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Aldehído Reductasa/metabolismo , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Aldehído Reductasa/genética , Animales , Cadherinas/genética , Cadherinas/metabolismo , Células Cultivadas , Regulación hacia Abajo , Células Epiteliales/citología , Transición Epitelial-Mesenquimal/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Cristalino/citología , Ratones , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismoRESUMEN
Aldose reductase (AR) in the lens plays an important role in the pathogenesis of diabetic cataract (DC) by contributing to osmotic and oxidative stress associated with accelerated glucose metabolism through the polyol pathway. Therefore, inhibition of AR in the lens may hold the key to prevent DC formation. Emodin, a bioactive compound isolated from plants, has been implicated as a therapy for diabetes. However, its inhibitory activity against AR remains unclear. Our results showed that emodin has good selectively inhibitory activity against AR (IC50 = 2.69 ± 0.90 µM) but not other aldo-keto reductases and is stable at 37 °C for at least 7 days. Enzyme kinetic studies demonstrated an uncompetitive inhibition against AR with a corresponding inhibition constant of 2.113 ± 0.095 µM. In in vivo studies, oral administration of emodin reduced the incidence and severity of morphological markers of cataract in lenses of AR transgenic mice. Computational modeling of the AR-NADP(+)-emodin ternary complex indicated that the 3-hydroxy group of emodin plays an essential role by interacting with Ser302 through hydrogen bonding in the specificity pocket of AR. All the findings above provide encouraging evidence for emodin as a potential therapeutic agent to prevent cataract in diabetic patients.
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Aldehído Reductasa/antagonistas & inhibidores , Catarata/tratamiento farmacológico , Complicaciones de la Diabetes/tratamiento farmacológico , Diabetes Mellitus Experimental/tratamiento farmacológico , Emodina/farmacología , Aldehído Reductasa/metabolismo , Aldo-Ceto Reductasas , Animales , Catarata/prevención & control , Humanos , Cristalino/enzimología , Ratones Transgénicos , Estructura Molecular , Estrés Oxidativo , Relación Estructura-ActividadRESUMEN
Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.
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Aldehído Reductasa/inmunología , Inflamación/inmunología , Microglía/inmunología , Retina/inmunología , Aldehído Reductasa/genética , Animales , Células Cultivadas , Citocinas/inmunología , Eliminación de Gen , Inflamación/genética , Lipopolisacáridos/inmunología , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Retina/citología , Retina/metabolismo , Salmonella typhimurium/inmunología , Regulación hacia ArribaRESUMEN
Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the lens to disturbances in signaling through the ERK and JNK pathways and thereby alter the balance of cell growth and apoptosis that is critical to lens transparency and homeostasis.
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Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Catarata/enzimología , Animales , Apoptosis , Catarata/etiología , Catarata/genética , Catarata/metabolismo , Complicaciones de la Diabetes/genética , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Glucosa/genética , Glucosa/metabolismo , Humanos , Hiperglucemia/genética , Hiperglucemia/metabolismo , Insulina/genética , Insulina/metabolismo , Cristalino/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Riesgo , Sorbitol/metabolismoRESUMEN
In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.
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Cristalinas/farmacología , Citoprotección/efectos de los fármacos , Calor , Cristalino/patología , Estrés Oxidativo/efectos de los fármacos , Cadena B de alfa-Cristalina/farmacología , Aldehído Reductasa/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Cristalinas/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/toxicidadRESUMEN
ß-Glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original two-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent, and selective therapeutic lead toward the treatment of diabetic eye disease.
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Aldehído Reductasa/antagonistas & inhibidores , Amidas/síntesis química , Inhibidores Enzimáticos/síntesis química , Glicósidos/síntesis química , Taninos Hidrolizables/química , Amidas/química , Amidas/farmacología , Diseño de Fármacos , Estabilidad de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicósidos/química , Glicósidos/farmacología , Humanos , Taninos Hidrolizables/síntesis químicaRESUMEN
We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent, with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations that did not inhibit NQO1 and were nontoxic to cells. Computation-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel with the isoalloxazine ring of FAD, and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition, including a 5-aminoalkylamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for the correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least 1 order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems.
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Indolquinonas/química , Modelos Moleculares , Quinona Reductasas/antagonistas & inhibidores , Quinona Reductasas/química , Alquilación , Aminación , Unión Competitiva , Dominio Catalítico , Sistema Libre de Células/enzimología , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Humanos , Indolquinonas/farmacología , Células K562 , Simulación de Dinámica Molecular , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH2). The formation of 17AAGH2 by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH2 were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH2 could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH2), reduction of 17AAG to 17AAGH2 by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH2. In addition, oxidation of 17AAGH2 could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH2 was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH2 with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH2, the active Hsp90 inhibitor derived from 17AAG.
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Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/farmacología , NAD(P)H Deshidrogenasa (Quinona)/fisiología , Neoplasias Pancreáticas/metabolismo , Superóxido Dismutasa/fisiología , Línea Celular Tumoral , Humanos , Neoplasias Pancreáticas/patologíaRESUMEN
Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis.
Asunto(s)
Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Neoplasias de la Boca/virología , Análisis por Matrices de Proteínas/métodos , Proteínas Virales/genética , Adulto , Anciano , Femenino , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , TaiwánRESUMEN
The indolequinone ES936 {5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione} was previously developed in our lab as an antitumor agent against pancreatic cancer. The objective of this study was to identify indolequinones with improved potency against pancreatic cancer and to define their mechanisms of action. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used in in vitro assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and clonogenic assays]; indolequinones displayed potent cytotoxicity against all three cell lines, and two specific classes of indolequinone were particularly potent agents. These indolequinones induced caspase-dependent apoptosis but no redox cycling or oxidative stress in MIA PaCa-2 and BxPC-3 cells. Selected indolequinones were also screened against the NCI-60 cell line panel and were found to be particularly effective against colon, renal, and melanoma cancer cells. A potential target of these indolequinones was identified as thioredoxin reductase. Indolequinones were found to be potent inhibitors of thioredoxin reductase activity both in pancreatic cancer cells and in cell-free systems. The mechanism of action of the indolequinones was shown to involve metabolic reduction, loss of a leaving group to generate a reactive electrophile resulting in alkylation of the selenocysteine residue in the active site of thioredoxin reductase. In vivo efficacy of the indolequinones was also tested in the MIA PaCa-2 pancreatic tumor xenograft in nude mice, and lead indolequinones demonstrated high efficacy and low toxicity. Inhibition of thioredoxin reductase represents a potential novel target in pancreatic cancer and may provide a biomarker of effect of lead indolequinones in this type of cancer.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Indolquinonas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Caspasas/fisiología , Línea Celular Tumoral , Sistema Libre de Células , Roturas del ADN de Cadena Simple , Femenino , Humanos , Ratones , Neoplasias Pancreáticas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Herpes simplex virus type 1 (HSV-1) infection is the most common cause of sporadic, fatal encephalitis, but current understanding of how the virus interacts with cellular factors to regulate disease progression is limited. Here, we show that HSV-1 infection induced the expression of the cellular transcription factor early growth response 1 (Egr-1) in a human neuronal cell line. Egr-1 increased viral replication by activating promoters of viral productive cycle genes through binding to its corresponding sequences in the viral promoters. Mouse studies confirmed that Egr-1 expression was enhanced in HSV-1-infected brains and that Egr-1 functions to promote viral replication in embryonic fibroblasts. Furthermore, Egr-1 deficiency or knockdown of Egr-1 by a DNA-based enzyme greatly reduced the mortality of HSV-1-infected mice by decreasing viral loads in tissues. This study provides what we believe is the first evidence that Egr-1 increases the mortality of HSV-1 encephalitis by enhancing viral replication. Moreover, blocking this cellular machinery exploited by the virus could prevent host mortality.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica , Herpes Simple/mortalidad , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/patogenicidad , Animales , Western Blotting , Tronco Encefálico/patología , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Herpes Simple/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Factores de Tiempo , Células Vero , Carga Viral , Replicación ViralRESUMEN
BACKGROUND AND PURPOSE: Agaricus blazei Murill has been reported to possess biological effects that include immunomodulatory and tumoricidal activities, but its potential effects have not been systematically analyzed in vivo. We evaluated the immunomodulatory effects of A. blazei in Balb/cByJ mice. METHODS: 160 male Balb/cByJ mice were divided into four groups and treated with various quantities of intragastric A. blazei extract or distilled water for 8 to 10 weeks. Nine parameters, relating to general immune function or adaptive immunity against immunogen chicken oval albumin, were determined. RESULTS: The results revealed that mice receiving A. blazei extract exhibited significantly greater serum immunoglobulin G levels, increased T-cell numbers in spleen, and elevated phagocytic capability compared with controls. Consumption of A. blazei was also associated with significant increases in oval albumin-specific serum immunoglobulin G level, delayed-type hypersensitivity, splenocyte proliferation rate, and tumor necrosis factor-alpha secretion by splenocytes. CONCLUSIONS: Consumption of A. blazei extract was associated with significant enhancement of seven out of nine immune functions tested. We conclude that A. blazei Murill possesses a wide range of immunomodulatory effects in vivo.
Asunto(s)
Agaricus/inmunología , Sistema Inmunológico/fisiopatología , Factores Inmunológicos , Animales , Hipersensibilidad Tardía/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Fagocitosis , Bazo/inmunología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have previously shown that a Taiwanese cohort of HIV-uninfected individuals was associated with the significantly elevated levels of serum beta-chemokines, macrophage inflammatory protein (MIP-1)-alpha and MIP-beta, and RANTES. In the present study, we report that the members of this cohort have significantly greater numbers of lower buoyant-density neutrophils in their blood, which leads to further investigation of the effects of beta-chemokines on neutrophils. By electron and confocal microscopic techniques and FACScan, the results demonstrated that MIP-1alpha, MIP-beta, and/or RANTES readily activated the cells to release a large quantity of alpha-defensins in vitro through the degranulation process, which was the cause of low-buoyant-density neutrophil production. The purified neutrophils underwent chemotaxis and increased phagocytic capability when beta-chemokines were present. Only when using all 3 neutralizing antibodies for CCR1, CCR3, and CCR5 could the chemotaxis of neutrophils be inhibited completely, suggesting that these receptors are involved in transducing activating signals. Because neutrophils are the most abundant white blood cells that can be activated simultaneously to release alpha-defensins and because these proteins are antiviral, including anti-HIV, our results support the hypothesis that in addition to beta-chemokines, the innate immunity of the cohort plays a role in inhibiting the transmission of HIV.