RESUMEN
Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD+). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD+-dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD+ metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Citocinas/genética , Glioblastoma/metabolismo , NAD/biosíntesis , Nicotinamida Fosforribosiltransferasa/genética , Tolerancia a Radiación , Transcripción Genética , Animales , Antineoplásicos/farmacología , Encéfalo/patología , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/radioterapia , Humanos , Ratones , Mutación , Trasplante de Neoplasias , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Células Madre/citologíaRESUMEN
Anti-vascular endothelial growth factor (anti-VEGF) antibodies are a promising new treatment for late time-to-onset radiation-induced necrosis (RN). We sought to evaluate and validate the response to anti-VEGF antibody in a mouse model of RN. Mice were irradiated with the Leksell Gamma Knife Perfexion™ and then treated with anti-VEGF antibody, beginning at post-irradiation (PIR) week 8. RN progression was monitored via anatomic and diffusion MRI from weeks 4-12 PIR. Standard histology, using haematoxylin and eosin (H&E), and immunohistochemistry staining were used to validate the response to treatment. After treatment, both post-contrast T1-weighted and T2-weighted image-derived lesion volumes decreased (P < 0.001), while the lesion volumes for the control group increased. The abnormally high apparent diffusion coefficient (ADC) for RN also returned to the ADC range for normal brain following treatment (P < 0.001). However, typical RN pathology was still present histologically. Large areas of focal calcification were observed in ~50% of treated mouse brains. Additionally, VEGF and hypoxia-inducible factor 1-alpha (HIF-1α) were continually upregulated in both the anti-VEGF and control groups. Despite improvements observed radiographically following anti-VEGF treatment, lesions were not completely resolved histologically. The subsequent calcification and the continued upregulation of VEGF and HIF-1α merit further preclinical/clinical investigation.
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Anticuerpos Monoclonales/farmacología , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Protectores contra Radiación/farmacología , Radiocirugia , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/efectos de la radiación , Lesiones Encefálicas/diagnóstico por imagen , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/etiología , Lesiones Encefálicas/patología , Calcinosis/diagnóstico por imagen , Calcinosis/tratamiento farmacológico , Calcinosis/etiología , Calcinosis/patología , Progresión de la Enfermedad , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Imagen por Resonancia Magnética , Ratones Endogámicos BALB C , Necrosis/diagnóstico por imagen , Necrosis/tratamiento farmacológico , Necrosis/etiología , Necrosis/patología , Traumatismos Experimentales por Radiación/diagnóstico por imagen , Traumatismos Experimentales por Radiación/patología , Distribución Aleatoria , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Pentose phosphate pathway (PPP) is a metabolic pathway that generates NADPH and pentose. PPP genes have been reported to be primarily or secondarily upregulated in many cancers. We aimed to study the general alteration of PPP in acute myelogenous leukemia (AML). We performed data mining and analysis of the Cancer Genome Atlas (TCGA) AML dataset for genetic alteration of the PPP gene set. In vitro studies including proliferation, migration, and invasion assays, together with metabolite consumption and oxidation assays, were performed. PPP genes were upregulated in 61 % of patients with AML. The majority of altered cases were expression changes measured by RNA sequencing. Expressions of critical PPP genes such as G6PD, PFKL, PFKP, and PGLS were consistently upregulated in all altered cases. Altered PPP is not associated with survival or disease relapse. PPP inhibition using 6-aminonicotinamide (6AN) increases glucose oxidative metabolism in AML. 6AN decreased the glucose oxidation and increased fatty acid oxidation. Here, we showed that PPP inhibition increased glucose oxidative metabolism in AML. PPP inhibition suppressed growth, migration, and invasion of AML, but not colony formation. PPP plays an important role in AML. Our results could contribute to the development of novel targeted treatment.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Vía de Pentosa Fosfato , 6-Aminonicotinamida/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Variación Genética , Glucosa/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Oxidación-Reducción/efectos de los fármacos , PronósticoRESUMEN
OBJECTIVES: Aspirin (ASA) has been frequently used for thromboprophylaxis in patients with multiple myeloma (MM) when treated with thalidomide or lenalidomide. Despite the well-recognized chemopreventive role of ASA in some solid tumors particularly for colon cancer, whether ASA displays the antimyeloma activity remains unclear. METHODS: MM1.S and RPMI-8226 cell lines harboring K-Ras and N-Ras mutation, respectively, were treated with various concentrations of ASA for different hours. The cell proliferation and apoptosis were performed to explore the effects of ASA on myeloma. Then, the exact mechanisms governing ASA's antimyeloma were explored by qRT-PCR and Western blot. Also, the effect of ASA on tumor growth was observed in NOD/SCID mice bearing myeloma xenografts. RESULTS: ASA of 0-10 mm concentration inhibits proliferation MM1.S and RPMI-8226 cells in time- and dose-dependent manner. The myeloma cells exposed to ASA treatment displayed concentration-dependent apoptosis, which was closely associated with activation of caspases, upregulation of Bax, and downregulation of Bcl-2 and VEGF. Study in vivo revealed that ASA administration retarded the tumor growth accompanying the survival time of mice bearing myeloma xenografts. CONCLUSIONS: ASA exerted antiproliferative and pro-apoptotic action in myeloma cells in vitro and delayed the growth of human myeloma cells in vivo. The underlying mechanisms were ascribed to regulation of Bcl-2 and Bax and suppression of VEGF.
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Apoptosis/efectos de los fármacos , Aspirina/farmacología , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Aspirina/administración & dosificación , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/genéticaRESUMEN
Purpose: Distinguishing radiation necrosis (RN) from recurrent tumor remains a vexing clinical problem with important health-care consequences for neuro-oncology patients. Here, mouse models of pure tumor, pure RN, and admixed RN/tumor are employed to evaluate hydrogen (1H) and deuterium (2H) magnetic resonance methods for distinguishing RN vs. tumor. Furthermore, proof-of-principle, range-finding deuterium (2H) metabolic magnetic resonance is employed to assess glycolytic signatures distinguishing RN vs. tumor. Materials and Methods: A pipeline of common quantitative 1H MRI contrasts, including an improved magnetization transfer ratio (MTR) sequence, and 2H magnetic resonance spectroscopy (MRS) following administration of 2H-labeled glucose, was applied to C57BL/6 mouse models of the following: (i) late time-to-onset RN, occurring 4-5 weeks post focal 50-Gy (50% isodose) Gamma Knife irradiation to the left cerebral hemisphere, (ii) glioblastoma, growing ~18-24 days post implantation of 50,000 mouse GL261 tumor cells into the left cerebral hemisphere, and (iii) mixed model, with GL261 tumor growing within a region of radiation necrosis (1H MRI only). Control C57BL/6 mice were also examined by 2H metabolic magnetic resonance. Results: Differences in quantitative 1H MRI parametric values of R1, R2, ADC, and MTR comparing pure tumor vs. pure RN were all highly statistically significant. Differences in these parameter values and DCEAUC for tumor vs. RN in the mixed model (tumor growing in an RN background) are also all significant, demonstrating that these contrasts-in particular, MTR-can effectively distinguish tumor vs. RN. Additionally, quantitative 2H MRS showed a highly statistically significant dominance of aerobic glycolysis (glucose â lactate; fermentation, Warburg effect) in the tumor vs. oxidative respiration (glucose â TCA cycle) in the RN and control brain. Conclusions: These findings, employing a pipeline of quantitative 1H MRI contrasts and 2H MRS following administration of 2H-labeled glucose, suggest a pathway for substantially improving the discrimination of tumor vs. RN in the clinic.
RESUMEN
Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. We recently described the ability of the small molecule, KCC009, a tissue transglutaminase (TG2) inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with nucleophilic cysteine thiol residues in the active sites of proteins that have an affinity to the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesive complexes and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Glioblastoma/patología , Isoxazoles/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Glioblastoma/tratamiento farmacológico , Humanos , Etiquetado Corte-Fin in Situ , Isoxazoles/química , Ratones , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
PURPOSE: Clinical evidence suggests radiation induces changes in the brain microenvironment that affect subsequent response to treatment. This study investigates the effect of previous radiation, delivered six weeks prior to orthotopic tumor implantation, on subsequent tumor growth and therapeutic response to anti-PD-L1 therapy in an intracranial mouse model, termed the Radiation Induced Immunosuppressive Microenvironment (RI2M) model. METHOD AND MATERIALS: C57Bl/6 mice received focal (hemispheric) single-fraction, 30-Gy radiation using the Leksell GammaKnife® Perfexion™, a dose that does not produce frank/gross radiation necrosis. Non-irradiated GL261 glioblastoma tumor cells were implanted six weeks later into the irradiated hemisphere. Lesion volume was measured longitudinally by in vivo MRI. In a separate experiment, tumors were implanted into either previously irradiated (30 Gy) or non-irradiated mouse brain, mice were treated with anti-PD-L1 antibody, and Kaplan-Meier survival curves were constructed. Mouse brains were assessed by conventional hematoxylin and eosin (H&E) staining, IBA-1 staining, which detects activated microglia and macrophages, and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Tumors in previously irradiated brain display aggressive, invasive growth, characterized by viable tumor and large regions of hemorrhage and necrosis. Mice challenged intracranially with GL261 six weeks after prior intracranial irradiation are unresponsive to anti-PD-L1 therapy. K-M curves demonstrate a statistically significant difference in survival for tumor-bearing mice treated with anti-PD-L1 antibody between RI2M vs. non-irradiated mice. The most prominent immunologic change in the post-irradiated brain parenchyma is an increased frequency of activated microglia. CONCLUSIONS: The RI2M model focuses on the persisting (weeks-to-months) impact of radiation applied to normal, control-state brain on the growth characteristics and immunotherapy response of subsequently implanted tumor. GL261 tumors growing in the RI2M grew markedly more aggressively, with tumor cells admixed with regions of hemorrhage and necrosis, and showed a dramatic loss of response to anti-PD-L1 therapy compared to tumors in non-irradiated brain. IHC and FACS analyses demonstrate increased frequency of activated microglia, which correlates with loss of sensitivity to checkpoint immunotherapy. Given that standard-of-care for primary brain tumor following resection includes concurrent radiation and chemotherapy, these striking observations strongly motivate detailed assessment of the late effects of the RI2M on tumor growth and therapeutic efficacy.
RESUMEN
OBJECTIVE: To investigate the incidence of JAK2V617F gene point mutation in patients with myeloproliferatives diseases (MPD) and its clinical significance. METHODS: Genomic DNA from bone marrow and peripheral blood cells were extracted from 68 patients with MPD. Allele specific polymerase chain reaction was used to amplify the exon 12 of JAK2 gene which harbours V617F mutation. The PCR products were identified by DNA sequencing. JAK2V617F gene point mutation and its impact on peripheral blood cells were analyzed. RESULTS: The incidence of JAK2V617F mutation in 68 patients with MPD was 65.28 %. The positive rate of JAK2V617F point mutation was 77.77 % in patients with PV (36/59), 56.52 % in patients with ET (23/59) and 44.44 % in patients with IMF (4/9). In all groups, the incidence of JAK2V617F point mutation in bone marrow and peripheral blood were equal. Patients with JAK2V617F mutation in PV group had higher counts of white blood cell and hemoglobin in peripheral blood than patients without JAK2V617F point mutation (P <0.05). Patients with JAK2V617F mutation in ET group had higher counts of white blood cell than those without JAK2V617F mutation (P <0.05); there was no significant difference in platelet count. CONCLUSION: JAK2V617F point mutation can affect the hematologic features, which may be of diagnostic value for MDP with negative BCR-ABL gene.
Asunto(s)
Sustitución de Aminoácidos , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/genética , Mutación Puntual , Adolescente , Adulto , Anciano , Secuencia de Bases , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Trastornos Mieloproliferativos/enzimología , Adulto JovenRESUMEN
PURPOSE: Glioblastoma (GBM) remains incurable, despite state-of-the-art treatment involving surgical resection, chemotherapy, and radiation. GBM invariably recurs as a highly invasive and aggressive phenotype, with the majority of recurrences within the radiation therapy treatment field. Although a large body of literature reporting on primary GBM exists, comprehensive studies of how prior irradiation alters recurrent tumor growth are lacking. An animal model that replicates the delayed effects of radiation therapy on the brain microenvironment, and its impact on the development of recurrent GBM, would be a significant advance. METHODS AND MATERIALS: Cohorts of mice received a single fraction of 0, 20, 30, or 40 Gy Gamma Knife irradiation. Naïve, nonirradiated mouse GBM tumor cells were implanted into the ipsilateral hemisphere 6 weeks postirradiation. Tumor growth was measured by magnetic resonance imaging, and animal survival was assessed by monitoring weight loss. Magnetic resonance imaging results were supported by hemotoxylin and eosin histology. RESULTS: Tumorous lesions generated from orthotopic implantation of nonirradiated mouse GBM tumor cells into irradiated mouse brain grew far more aggressively and invasively than implantation of these same cells into nonirradiated brain. Lesions in irradiated brain tissue were significantly larger, more necrotic, and more vascular than those in control animals with increased invasiveness of tumor cells in the periphery, consistent with the histologic features commonly observed in recurrent high-grade tumors in patients. CONCLUSIONS: Irradiation of normal brain primes the targeted cellular microenvironment for aggressive tumor growth when naïve (not previously irradiated) cancer cells are subsequently introduced. The resultant growth pattern is similar to the highly aggressive pattern of tumor regrowth observed clinically after therapeutic radiation therapy. The mouse model offers an avenue for determining the cellular and molecular basis for the aggressiveness of recurrent GBM.
Asunto(s)
Neoplasias Encefálicas/radioterapia , Encéfalo/efectos de la radiación , Microambiente Celular/efectos de la radiación , Glioblastoma/radioterapia , Animales , Encéfalo/patología , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de la radiación , Femenino , Glioblastoma/patología , Ratones , Ratones Endogámicos BALB C , Invasividad NeoplásicaRESUMEN
PURPOSE: There is mounting evidence that, in addition to angiogenesis, hypoxia-induced inflammation via the hypoxia-inducible factor 1α (HIF-1α)-CXC chemokine receptor 4 (CXCR4) pathway may contribute to the pathogenesis of late-onset, irradiation-induced necrosis. This study investigates the mitigative efficacy of an HIF-1α inhibitor, topotecan, and a CXCR4 antagonist, AMD3100, on the development of radiation necrosis (RN) in an intracranial mouse model. METHODS AND MATERIALS: Mice received a single-fraction, 50-Gy dose of hemispheric irradiation from the Leksell Gamma Knife Perfexion and were then treated with either topotecan, an HIF-1α inhibitor, from 1 to 12 weeks after irradiation, or AMD3100, a CXCR4 antagonist, from 4 to 12 weeks after irradiation. The onset and progression of RN were monitored longitudinally via noninvasive, in vivo magnetic resonance imaging (MRI) from 4 to 12 weeks after irradiation. Conventional hematoxylin-eosin staining and immunohistochemistry staining were performed to evaluate the treatment response. RESULTS: The progression of brain RN was significantly mitigated for mice treated with either topotecan or AMD3100 compared with control animals. MRI-derived lesion volumes were significantly smaller for both of the treated groups, and histologic findings correlated well with the MRI data. By hematoxylin-eosin staining, both treated groups demonstrated reduced irradiation-induced tissue damage compared with controls. Furthermore, immunohistochemistry results revealed that expression levels of vascular endothelial growth factor, CXC chemokine ligand 12, CD68, CD3, and tumor necrosis factor α in the lesion area were significantly lower in treated (topotecan or AMD3100) brains versus control brains, while ionized calcium-binding adapter molecule 1 (Iba1) and HIF-1α expression was similar, though somewhat reduced. CXCR4 expression was reduced only in topotecan-treated mice, while interleukin 6 expression was unaffected by either topotecan or AMD3100. CONCLUSIONS: By reducing inflammation, both topotecan and AMD3100 can, independently, mitigate the development of RN in the mouse brain. When combined with first-line, antiangiogenic treatment, anti-inflammation therapy may provide an adjuvant therapeutic strategy for clinical, postirradiation management of tumors, with additional benefits in the mitigation of RN development.
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Encéfalo/patología , Compuestos Heterocíclicos/uso terapéutico , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Traumatismos Experimentales por Radiación/prevención & control , Receptores CXCR4/antagonistas & inhibidores , Topotecan/uso terapéutico , Animales , Bencilaminas , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de la radiación , Ciclamas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Necrosis/diagnóstico por imagen , Necrosis/etiología , Necrosis/patología , Necrosis/prevención & control , Traumatismos Experimentales por Radiación/diagnóstico por imagen , Traumatismos Experimentales por Radiación/patologíaRESUMEN
In our previous study, it was found that aspirin (ASA) exerted antimyeloma actions in vivo and in vitro. The resistance to bortezomib (BTZ) in multiple myeloma (MM) is partly due to AKT activation and the upregulation of survivin induced by BTZ, which are the targets of ASA in gastric and ovarian cancer, respectively. Thus, the present study investigated the interaction between ASA and BTZ in MM and further clarified the underlying mechanisms. MM1.S and RPMI-8226 cell lines harboring the N- and K-Ras mutations, respectively, were treated with 2.5 mM ASA, 10 nM BTZ and ASA+BTZ for different durations. The proliferation and apoptosis of the cells were determined, and the underlying mechanisms governing the interaction of ASA and BTZ were examined in the MM cells. Treatment with ASA+BTZ caused higher rates of proliferative inhibition and apoptosis in the MM1.S and RPMI-8226 cells in time-dependent manner, compared with either agent alone. A drug interaction assay revealed the additive effect of ASA and BTZ on the myeloma cells. ASA alone inhibited the levels of phosphorylated AKT (p-AKT) and survivin, whereas BTZ alone augmented the levels of p-AKT and survivin. Of note, ASA markedly decreased the upregulation of p-AKT and survivin induced by BTZ. Treatment with ASA+BTZ significantly suppressed the level of Bcl-2, compared with either agent alone. ASA may potentiate the antimyeloma activity of BTZ against myeloma cells via suppression of AKT phosphorylation, survivin and Bcl-2, indicating the potential of ASA+BTZ in treating MM, particularly for cases of BTZ-refractory/relapsed MM.
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Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes.
Asunto(s)
Apoptosis/fisiología , Proteínas del Ojo/fisiología , Empalme del ARN/fisiología , Retina/citología , Rodopsina/genética , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/genética , Inhibidores de Caspasas , Caspasas/fisiología , Células Cultivadas , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Humanos , Ratones , Mutación , Neuronas/citología , Neuronas/metabolismo , Empalme del ARN/genética , Retina/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Rodopsina/biosíntesisRESUMEN
3-halo-4,5-dihydroisoxazoles are attractive warheads for the selective inhibition of nucleophilic active sites in biological systems. A series of 3-bromo-4,5-dihydroisoxazole compounds were prepared and tested for their ability to irreversibly inhibit human transglutaminase 2 (TG2), an enzyme that plays an important role in the pathogenesis of diverse disorders including Celiac Sprue and certain types of cancers. Several compounds showed high specificity for human TG2 (k(inh)/K(I) > 2000 min(-1)M(-1)) but essentially no reactivity (k < 1 min(-1)M(-1)) toward physiological thiols such as glutathione. The pharmacokinetic and pharmacodynamic properties of a prototype dihydroisoxazole inhibitor, 1b, were evaluated; in mice the compound showed good oral bioavailability, short serum half-life and efficient TG2 inhibition in small intestinal tissue, and low toxicity. It also showed excellent synergism with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine) against refractory glioblastoma tumors in mice. A fluorescent dihydroisoxazole inhibitor 5 facilitated microscopic visualization of TG2 endocytosis from the extracellular surface of HCT-116 cells. Together, these findings demonstrate the promise of dihydroisoxazole compounds as probes for the biology of TG2 and its role in human disease.
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Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/antagonistas & inhibidores , Isoxazoles/química , Isoxazoles/farmacología , Transglutaminasas/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Carmustina/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacocinética , Proteínas de Unión al GTP/biosíntesis , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Isoxazoles/farmacocinética , Ratones , Ratones Endogámicos C3H , Microscopía Confocal , Proteína Glutamina Gamma Glutamiltransferasa 2 , Relación Estructura-Actividad , Transglutaminasas/biosíntesisRESUMEN
Tissue transglutaminase 2 belongs to a family of transglutaminase proteins that confers mechanical resistance from proteolysis and stabilizes proteins. Transglutaminase 2 promotes transamidation between glutamine and lysine residues with the formation of covalent linkages between proteins. Transglutaminase 2 also interacts and forms complexes with proteins important in extracellular matrix organization and cellular adhesion. We have identified the novel finding that treatment of glioblastoma cells with transglutaminase 2 inhibitors promotes cell death and enhances sensitivity to chemotherapy. Treatment with either the competitive transglutaminase 2 inhibitor, monodansylcadaverine, or with highly specific small-molecule transglutaminase 2 inhibitors, KCA075 or KCC009, results in induction of apoptosis in glioblastoma cells. Treatment with these transglutaminase 2 inhibitors resulted in markedly decreased levels of the prosurvival protein, phosphorylated Akt, and its downstream targets. These changes promote a proapoptotic profile with altered levels of multiple intracellular proteins that determine cell survival. These changes include decreased levels of the antiapoptotic proteins, survivin, phosphorylated Bad, and phosphorylated glycogen synthetase kinase 3beta (GSK-3beta), and increased levels of the proapoptotic BH3-only protein, Bim. In vivo studies with s.c. murine DBT glioblastoma tumors treated with transglutaminase 2 inhibitors combined with the chemotherapeutic agent, N-N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), decreased tumor size based on weight by 50% compared with those treated with BCNU alone. Groups treated with transglutaminase 2 inhibitors showed an increased incidence of apoptosis determined with deoxynucleotidyl transferase-mediated biotin nick-end labeling staining. These studies identify inhibition of transglutaminase 2 as a potential target to enhance cell death and chemosensitivity in glioblastomas.
Asunto(s)
Muerte Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/antagonistas & inhibidores , Glioblastoma/patología , Transglutaminasas/antagonistas & inhibidores , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Compuestos Azo/síntesis química , Compuestos Azo/farmacología , Proteína 11 Similar a Bcl2 , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Cadaverina/análogos & derivados , Cadaverina/farmacología , Carmustina/farmacología , Resistencia a Antineoplásicos , Glioblastoma/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas Inhibidoras de la Apoptosis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilación , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Survivin , Células Tumorales CultivadasRESUMEN
Two [(18)F]fluoroalkyl substituted amino acids differing only by the presence or absence of a methyl group on the α-carbon, (S)-2-amino-7-[(18)F]fluoro-2-methylheptanoic acid ((S)-[(18)F]FAMHep, (S)-[(18)F]14) and (S)-2-amino-7-[(18)F]fluoroheptanoic acid ((S)-[(18)F]FAHep, (S)-[(18)F]15), were developed for brain tumor imaging and compared to the well-established system L amino acid tracer, O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]FET), in the delayed brain tumor (DBT) mouse model of high-grade glioma. Cell uptake, biodistribution, and PET/CT imaging studies showed differences in amino acid transport of these tracer by DBT cells. Recognition of (S)-[(18)F]15 but not (S)-[(18)F]14 by system L amino acid transporters led to approximately 8-10-fold higher uptake of the α-hydrogen substituted analogue (S)-[(18)F]15 in normal brain. (S)-[(18)F]15 had imaging properties similar to those of (S)-[(18)F]FET in the DBT tumor model while (S)-[(18)F]14 afforded higher tumor to brain ratios due to much lower uptake by normal brain. These results have important implications for the future development of α-alkyl and α,α-dialkyl substituted amino acids for brain tumor imaging.
Asunto(s)
Aminoácidos Neutros/farmacocinética , Aminoácidos/farmacocinética , Neoplasias Encefálicas/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Glioma/diagnóstico por imagen , Hidrógeno/química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Sustitución de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Aminoácidos Neutros/química , Aminoácidos Neutros/metabolismo , Animales , Transporte Biológico , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Radioisótopos de Flúor/química , Radioisótopos de Flúor/metabolismo , Radioisótopos de Flúor/farmacocinética , Glioma/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos BALB C , Radiofármacos/química , Radiofármacos/metabolismo , Distribución Tisular , Tirosina/análogos & derivadosRESUMEN
Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Vía de Pentosa Fosfato/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , 6-Aminonicotinamida/farmacología , Afatinib , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinib , Glucosa/metabolismo , Humanos , Metaboloma/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Oxígeno/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas ras/metabolismoRESUMEN
The novel compound, (S)-amino-2-methyl-4-[(76)Br]bromo-3-(E)-butenoic acid (BrVAIB, [(76)Br]5), was characterized against the known system A tracer, IVAIB ([(123)I]8). [(76)Br]5 was prepared in a 51% ± 19% radiochemical yield with high radiochemical purity (≥98%). The biological properties of [(76)Br]5 were compared with those of [(123)I]8. Results showed that [(76)Br]5 undergoes mixed amino acid transport by system A and system L transport, while [(123)I]8 had less uptake by system L. [(76)Br]5 demonstrated higher uptake than [(123)I]8 in DBT tumors 1 h after injection (3.7 ± 0.4% ID/g vs 1.5 ± 0.3% ID/g) and also showed higher uptake vs [(123)I]8 in normal brain. Small animal PET studies with [(76)Br]5 demonstrated good tumor visualization of intracranial DBTs up to 24 h with clearance from normal tissues. These results indicate that [(76)Br]5 is a promising PET tracer for brain tumor imaging and lead compound for a mixed system A and system L transport substrate.
Asunto(s)
Neoplasias Encefálicas/diagnóstico , Encéfalo/patología , Radioisótopos de Bromo/química , Crotonatos/química , Glioma/diagnóstico , Tomografía de Emisión de Positrones/métodos , Alanina/análogos & derivados , Alanina/farmacocinética , Aminación , Animales , Transporte Biológico , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Radioisótopos de Bromo/farmacocinética , Crotonatos/síntesis química , Crotonatos/farmacocinética , Glioma/metabolismo , Glioma/patología , Radioisótopos de Yodo/farmacocinética , Masculino , Ratones Endogámicos BALB C , Radiofármacos/síntesis química , Distribución TisularRESUMEN
A novel (18)F-labeled α,α-disubstituted amino acid-based tracer, 2-amino-5-[(18)F]fluoro-2-methylpentanoic acid ([(18)F]FAMPe), has been developed for brain tumor imaging with a longer alkyl side chain than previously reported compounds to increase brain availability via system L amino acid transport. Both enantiomers of [(18)F]FAMPe were obtained in good radiochemical yield (24-52% n = 8) and high radiochemical purity (>99%). In vitro uptake assays in mouse DBT gliomas cells revealed that (S)-[(18)F]FAMPe enters cells partly via sodium-independent system L transporters and also via other nonsystem A transport systems including transporters that recognize glutamine. Biodistribution and small animal PET/CT studies in the mouse DBT model of glioblastoma showed that both (R)- and (S)-[(18)F]FAMPe have good tumor imaging properties with the (S)-enantiomer providing higher tumor uptake and tumor to brain ratios. Comparison of the SUVs showed that (S)-[(18)F]FAMPe had higher tumor to brain ratios compared to (S)-[(18)F]FET, a well-established system L substrate.
Asunto(s)
Aminoácidos de Cadena Ramificada/química , Aminoácidos Neutros/química , Neoplasias Encefálicas/diagnóstico por imagen , Glioma/diagnóstico por imagen , Ácidos Pentanoicos/química , Radiofármacos/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos de Cadena Ramificada/síntesis química , Aminoácidos de Cadena Ramificada/farmacología , Aminoácidos Neutros/síntesis química , Aminoácidos Neutros/farmacología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Radioisótopos de Flúor , Glioma/metabolismo , Masculino , Ratones Endogámicos BALB C , Ácidos Pentanoicos/síntesis química , Ácidos Pentanoicos/farmacocinética , Tomografía de Emisión de Positrones , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Estereoisomerismo , Distribución TisularRESUMEN
BACKGROUND: Recently, we characterized a Gamma Knife® radiation necrosis mouse model with various magnetic resonance imaging (MRI) protocols to identify biomarkers useful in differentiation from tumors. Though the irradiation was focal to one hemisphere, a contralateral injury was observed that appeared to be localized in the white matter only. Interestingly, this injury was identifiable in T2-weighted images, apparent diffusion coefficient (ADC), and magnetization transfer ratio (MTR) maps, but not on post-contrast T1-weighted images. This observation of edema independent of vascular changes is akin to the perilesional edema seen in clinical radiation necrosis. FINDINGS: The pathology underlying the observed white-matter MRI changes was explored by performing immunohistochemistry for healthy axons and myelin. The presence of both healthy axons and myelin was reduced in the contralateral white-matter lesion. CONCLUSIONS: Based on our immunohistochemical findings, the contralateral white-matter injury is most likely due to axonal degeneration.