RESUMEN
PURPOSE: To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. METHODS AND MATERIALS: Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. RESULTS: Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. CONCLUSIONS: These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.
Asunto(s)
Barbitúricos/farmacología , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de los fármacos , Indoles/farmacología , Terapia Molecular Dirigida/métodos , Proteínas de Neoplasias/efectos de los fármacos , Proteínas Nucleares/efectos de los fármacos , Tolerancia a Radiación , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Fibroblastos/química , Fibroblastos/efectos de la radiación , Histonas/análisis , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/radioterapia , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/fisiología , Proteínas Nucleares/análisis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/fisiología , Nucleofosmina , Recombinasa Rad51/análisis , Análisis de Matrices Tisulares , Células Tumorales CultivadasRESUMEN
Targeting tumor vasculature represents a rational strategy for controlling cancer. (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (denoted VJ115) is a novel chemical entity that inhibits the enzyme ENOX1, a NADH oxidase. Genetic and small molecule inhibition of ENOX1 inhibits endothelial cell tubule formation and tumor-mediated neo-angiogenesis. Inhibition of ENOX1 radiosensitizes tumor vasculature, a consequence of enhanced apoptosis. However, the molecular mechanisms underlying these observations are not well understood. Herein, we mechanistically link ENOX1-mediated regulation of cellular NADH concentrations with proteomics profiling of endothelial cell protein expression following exposure to VJ115. Pathway Studios network analysis of potential effector molecules identified by the proteomics profiling indicated that a VJ115 exposure capable of altering intracellular NADH concentrations impacted proteins involved in cytoskeletal reorganization. The analysis was validated using RT-PCR and immunoblotting of selected proteins. RNAi knockdown of ENOX1 was shown to suppress expression of stathmin and lamin A/C, proteins identified by the proteomics analysis to be suppressed upon VJ115 exposure. These data support the hypothesis that VJ115 inhibition of ENOX1 can impact expression of proteins involved in cytoskeletal reorganization and support a hypothesis in which ENOX1 activity links elevated cellular NADH concentrations with cytoskeletal reorganization and angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Proteínas del Citoesqueleto/metabolismo , Indoles/farmacología , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Quinuclidinas/farmacología , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , NAD/metabolismo , ProteómicaRESUMEN
PURPOSE: Radiation therapy continues to be an important therapeutic strategy for providing definitive local/regional control of human cancer. However, oncogenes that harbor driver mutations and/or amplifications can compromise therapeutic efficacy. Thus, there is a need for novel approaches that enhance the DNA damage produced by ionizing radiation. EXPERIMENTAL DESIGN: A forward chemical genetic approach coupled with cell-based phenotypic screening of several tumor cell lines was used to identify a novel chemical entity (NCE) that functioned as a radiation sensitizer. Proteomics, comet assays, confocal microscopy, and immunoblotting were used to identify the biological target. RESULTS: The screening process identified a 5-((N-benzyl-1H-indol-3-yl)-methylene)pyrimidine-2,4,6(1H,3H,5H)trione as an NCE that radiosensitized cancer cells expressing amplified and/or mutated RAS, ErbB, PIK3CA, and/or BRAF oncogenes. Affinity-based solid-phase resin capture followed by liquid chromatography/tandem mass spectrometry identified the chaperone nucleophosmin (NPM) as the NCE target. SiRNA suppression of NPM abrogated radiosensitization by the NCE. Confocal microscopy showed that the NCE inhibited NPM shuttling to radiation-induced DNA damage repair foci, and the analysis of comet assays indicated a diminished rate of DNA double-strand break repair. CONCLUSION: These data support the hypothesis that inhibition of DNA repair due to inhibition of NPM shuttling increases the efficacy of DNA-damaging therapeutic strategies.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Barbitúricos/farmacología , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Humanos , Indoles/farmacología , Ratones , Ratones Desnudos , Chaperonas Moleculares/metabolismo , Terapia Molecular Dirigida , Neoplasias/metabolismo , Nucleofosmina , Tolerancia a Radiación/efectos de los fármacos , Distribución AleatoriaRESUMEN
There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.
Asunto(s)
Endotelio Vascular/citología , Neovascularización Patológica/tratamiento farmacológico , Proteína Disulfuro Reductasa (Glutatión)/antagonistas & inhibidores , Quinuclidinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Humanos , Indoles , Proteínas de la Membrana/antagonistas & inhibidores , Neoplasias/irrigación sanguínea , Neoplasias/terapia , Neovascularización Patológica/radioterapia , Quinuclidinas/uso terapéuticoRESUMEN
The transcription factor nuclear factor erythroid-derived 2-related factor 2 (Nrf2) regulates induction of an extensive cellular stress response network when complexed with the cAMP-responsive element binding protein (CBP) at antioxidant response elements (ARE) located in the promoter region of target genes. Activating transcription factor 3 (ATF3) can repress Nrf2-mediated signaling in a manner that is not well understood. Here, we show that ATF3-mediated suppression is a consequence of direct ATF3-Nrf2 protein-protein interactions that result in displacement of CBP from the ARE. This work establishes ATF3 as a novel repressor of the Nrf2-directed stress response pathway.
Asunto(s)
Factor de Transcripción Activador 3/fisiología , Regulación de la Expresión Génica , Factor 2 Relacionado con NF-E2/fisiología , Estrés Fisiológico/genética , Factor de Transcripción Activador 3/antagonistas & inhibidores , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Animales , Antioxidantes/farmacología , Células Cultivadas , Ratones , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica , ARN Interferente Pequeño/farmacología , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Elementos de Respuesta/efectos de los fármacos , Transducción de Señal/genética , TransfecciónRESUMEN
Use of ionizing radiation is essential for the management of many human cancers, and therapeutic hyperthermia has been identified as a potent radiosensitizer. Radiation therapy combined with adjuvant hyperthermia represents a potential tool to provide outstanding local-regional control for refractory disease. (Z)-(+/-)-2-(N-Benzylindol-3-ylmethylene)quinuclidin-3-ol (2) and (Z)-(+/-)-2-(N-benzenesulfonylindol-3-ylmethylene)quinuclidin-3-ol (4) were initially identified as potent thermal sensitizers that could lower the threshold needed for thermal sensitivity to radiation treatment. To define the structural requirements of the molecule that are essential for thermal sensitization, we have synthesized and evaluated a series of (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one (9), and (Z)-(+/-)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol (10) analogs that incorporate a variety of substituents in both the indole and N-benzyl moieties. These systematic structure-activity relationship (SAR) studies were designed to further the development and optimization of potential clinically useful thermal sensitizing agents. The most potent analog was compound 10 (R(1)=H, R(2)=4-Cl), which potently inhibited (93% inhibition at 50 microM) the growth of HT-29 cells after a 41 degrees C/2h exposure.
Asunto(s)
Quinuclidinas/síntesis química , Quinuclidinas/farmacología , Temperatura , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Estructura Molecular , Quinuclidinas/química , Fármacos Sensibilizantes a Radiaciones/síntesis química , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacología , Relación Estructura-ActividadRESUMEN
15-deoxy-Delta12,14-prostaglandin J2 (15-d-PGJ2) is a dehydration product of PGD2. This compound possesses a highly reactive polyunsaturated carbonyl moiety that is a substrate for Michael addition with thiol-containing biomolecules such as glutathione and cysteine residues on proteins. By reacting with glutathione and proteins, 15-d-PGJ2 is believed to exert potent biological activity. Despite the large number of publications that have ascribed bioactivity to this molecule, it is not known to what extent 15-d-PGJ2 is formed in vivo. Levels of free 15-d-PGJ2 measured in human biological fluids such as urine are low, and the biological importance of this compound has thus been questioned. Because of its reactivity, we hypothesized that 15-d-PGJ2 is present in vivo primarily as a Michael conjugate. Therefore, we undertook a detailed study of the metabolism of this compound in HepG2 cells that are known to metabolize other cyclopentenone eicosanoids. We report that HepG2 cells primarily convert 15-d-PGJ2 to a glutathione conjugate in which the carbonyl at C-11 is reduced to a hydroxyl. Subsequently, the glutathione portion of the molecule is hydrolyzed with loss of glutamic acid and glycine resulting in a cysteine conjugate. These findings confirm a general route for the metabolism of cyclopentenone eicosanoids in HepG2 cells and may pave the way for new insights regarding the formation of 15-d-PGJ2 in vivo.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Glutatión/metabolismo , Hepatocitos/metabolismo , Fase II de la Desintoxicación Metabólica , Prostaglandina D2/análogos & derivados , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Glutatión/análisis , Hepatocitos/efectos de los fármacos , Humanos , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(+/-)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation.
Asunto(s)
Neoplasias del Colon/terapia , Hipertermia Inducida/métodos , Indoles/química , Indoles/farmacología , Mitosis/fisiología , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Neoplasias del Colon/radioterapia , Proteínas de Unión al ADN/metabolismo , Células HCT116 , Factores de Transcripción del Choque Térmico , Humanos , Indoles/síntesis química , Mitosis/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/síntesis química , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Ácidos Grasos Omega-3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Proteínas de Ciclo Celular/química , Línea Celular , Proteínas Cullin/química , Ácidos Grasos Omega-3/química , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Estructura Molecular , Oxidación-Reducción , Estrés Oxidativo/genética , Activación TranscripcionalRESUMEN
Cyclopentenone isoprostanes (IsoPs), A(2)/J(2)-IsoPs, are one class of IsoPs formed via the free radical-initiated peroxidation of arachidonic acid. These compounds, which are structurally similar to cyclooxygenase-derived PGA(2) and PGJ(2), contain highly reactive alpha,beta-unsaturated carbonyl moieties. A(2)/J(2)-IsoPs are generated in vivo in humans esterified in glycerophospholipids. Unlike other classes of IsoPs, however, cyclopentenone IsoPs cannot be detected in the free form; we postulated that this might be due to their rapid adduction to various thiol-containing biomolecules via Michael addition. Recently, we reported that the A-ring IsoP, 15-A(2t)-IsoP, is efficiently conjugated with glutathione in vitro by certain human and rat glutathione transferases (GSTs), with the isozyme GSTA4-4 displaying the highest activity. Herein, we examined the metabolic disposition of 15-A(2t)-IsoP in HepG2 cells. We report that 15-A(2t)-IsoP is primarily metabolized by these cells via conjugation to glutathione. Within 6 h, approximately 60% of 15-A(2t)-IsoP added to HepG2 cells was present in the form of a water soluble conjugate(s). Structural characterization of the adduct(s) by liquid chromatography-tandem mass spectrometry revealed four major conjugates. These include the intact 15-A(2t)-IsoP-GSH conjugate, the GSH conjugate in which the carbonyl at C-9 of 15-A(2t)-IsoP is reduced, and the corresponding cysteine conjugates. These studies thus show that the primary pathway of metabolic disposition of endogenously derived cyclopentenone IsoPs occurs via conjugation with thiols.
Asunto(s)
Ciclopentanos/metabolismo , Glutatión/metabolismo , Peroxidación de Lípido , Prostaglandinas A/metabolismo , Cisteína/química , Cisteína/metabolismo , Glutatión/química , Humanos , Prostaglandinas A/química , Prostaglandinas A/farmacología , Espectrometría de Masa por Ionización de Electrospray , Factores de Tiempo , Tritio , Células Tumorales CultivadasRESUMEN
Glutamate cysteine ligase, the rate-limiting enzyme for the synthesis of glutathione, represents an important component of chemoprevention paradigms. GCLC and GCLM, the genes encoding glutamate cysteine ligase subunits, are induced by indoles, such as indomethacin. Novel functionalized indole analogues and other structurally related compounds were synthesized and used for a comparative structure analysis of GCLC induction. Use of mouse embryo fibroblasts null for Nrf2 (nuclear factor-erythroid 2p45-related transcription factor) and HepG2 cells overexpressing Keap1 demonstrated that indole analogue-mediated GCLC expression was regulated by Nrf2-Keap1 interactions. Indole analogues capable of inducing GCLC were found to increase NADPH oxidase activity. Indole analogues unable to induce GCLC did not increase oxidase activity. HepG2 cells transfected with FLAG/Keap1 were exposed to indomethacin, and the redox state of Keap1 cysteine residues was assessed. The data indicated that Keap1 exhibited several oxidation states that were sensitive to indomethacin treatment. These indomethacin-mediated changes in thiol oxidation states were suppressed by diphenyleneiodonium, a NADPH oxidase inhibitor. Diphenyleneiodonium also suppressed indole analogue-mediated increases in GCLC mRNA. In summary, the use of the indole analogues identified NADPH oxidase activity as a novel upstream activity regulating Nrf2/Keap1 signaling of GCLC, provided data supporting the hypothesis that Keap1 is a downstream effector for oxidase activity, and afforded in vivo data to support the hypothesis that Keap1 thiols can act as molecular sensors of reactive oxygen species. Finally, the comparative structure analysis suggests that 2-indol-3-yl-methylenequinuclidin-3-ols may represent a prototype for the development of novel chemopreventative agents able to activate Keap1/Nrf2 signaling.