RESUMEN
The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein ataxin-3, a deubiquitinase. Two major full-length isoforms of ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.
Asunto(s)
Ataxina-3/genética , Ataxina-3/toxicidad , Proteínas de Drosophila/genética , Proteínas de Drosophila/toxicidad , Enfermedad de Machado-Joseph/genética , Proteínas Represoras/genética , Proteínas Represoras/toxicidad , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Enfermedad de Machado-Joseph/fisiopatología , Isoformas de Proteínas/genética , Isoformas de Proteínas/toxicidadRESUMEN
In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome.
Asunto(s)
Antineoplásicos/química , Proteínas Inhibidoras de la Apoptosis/química , Nanopartículas/química , Proteínas Represoras/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis/farmacología , Proteínas Inhibidoras de la Apoptosis/toxicidad , Ratones , Mutación , Nanopartículas/toxicidad , Proteínas Represoras/farmacología , Proteínas Represoras/toxicidad , SurvivinRESUMEN
PURPOSE: To investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer (NSCLC) cells and the underlying mechanisms. METHODS: NSCLC cells (A549 and NCI-H460) transfected with EGFP plasmids containing HPV-16 E6 or E7 oncogene were treated with different concentrations of EGCG for 16 h. The effects of EGCG on angiogenesis in vitro and in vivo were observed. The expression of HIF-1α, p-Akt, and p-ERK1/2 proteins in NSCLC cells was analyzed by Western blot. The levels of HIF-1α mRNA in NSCLC cells were detected by real-time RT-PCR. The concentration of VEGF and IL-8 in the conditioned media was determined by ELISA. HIF-1α, VEGF, and CD31 expression in A549 xenografted tumors of nude mice was analyzed by immunohistochemistry. RESULTS: HPV-16 E6 and E7 oncoproteins HIF-1α-dependently promoted angiogenesis in vitro and in vivo, which was inhibited by EGCG. Mechanistically, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression in NSCLC cells. Additionally, 50 and 100 µmol/L of EGCG significantly reduced the secretion of VEGF and IL-8 proteins induced by HPV-16 E7 oncoprotein in NSCLC A549 cells. Meanwhile, HPV-16 E6 and E7 oncoproteins HIF-1α-dependently enhanced Akt activation in A549 cells, which was suppressed by EGCG. Furthermore, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α and HIF-1α-dependent VEGF and CD31 expression in A549 xenografted tumors. CONCLUSIONS: EGCG inhibited HPV-16 oncoprotein-induced angiogenesis conferred by NSCLC through the inhibition of HIF-1α protein expression and HIF-1α-dependent expression of VEGF, IL-8, and CD31 as well as activation of Akt, suggesting that HIF-1α may be a potential target of EGCG against HPV-related NSCLC angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Catequina/análogos & derivados , Papillomavirus Humano 16/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/prevención & control , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/toxicidad , Proteínas Oncogénicas/efectos de los fármacos , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/toxicidad , Animales , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Catequina/farmacología , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Interleucina-8/biosíntesis , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transfección , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.
Asunto(s)
Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Animales , División Celular , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/toxicidad , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/toxicidad , Transcripción Genética , Dedos de ZincRESUMEN
BACKGROUND: Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC. OBJECTIVES: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system. METHODS: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro. RESULTS: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes. CONCLUSIONS: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacología , Coagulación Intravascular Diseminada/sangre , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Represoras/metabolismo , Trombosis/sangre , Animales , Pruebas de Coagulación Sanguínea , Células Cultivadas , Coagulantes/toxicidad , Citocinas/sangre , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/inducido químicamente , Coagulación Intravascular Diseminada/metabolismo , Coagulación Intravascular Diseminada/patología , Activación Enzimática/efectos de los fármacos , Fibrina/metabolismo , Proteína HMGB1 , Hemólisis/efectos de los fármacos , Proteínas del Grupo de Alta Movilidad/farmacología , Proteínas del Grupo de Alta Movilidad/toxicidad , Humanos , Inflamación/sangre , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Proteína C/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/farmacología , Proteínas Represoras/toxicidad , Trombina , Tromboplastina/metabolismo , Trombosis/inducido químicamente , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
Cycloheximide, a reversible protein synthesis inhibitor, is thought to block DNA replication in normal cells by preventing synthesis of a labile protein. In animal systems, cycloheximide protects normal cells from cytotoxic S-phase specific agents, such as cytosine arabinoside (Ara-C). Malignant cells appear not to be susceptible to cycloheximide-induced cycle arrest and, subsequently, may not be protected from Ara-C cytotoxicity. The effect of cycloheximide on granulocyte/macrophage progenitors (CFU-GM) after in vitro Ara-C exposure was examined using normal human bone marrow, malignant progenitors from patients with chronic myelogenous leukemia (CML), and clonogenic cells from the human acute nonlymphocytic leukemia cell lines HL-60 and KG-1. Mononuclear or clonogenic cells were incubated for one hour with cycloheximide, followed by the addition, for three or 17 hours, of Ara-C before being plated in a methylcellulose culture system. CFU-GM survival was significantly increase if normal cells were treated with cycloheximide before Ara-C exposure. Similar cycloheximide pretreatment of CML progenitors and clonogenic HL-60 and KG-1 cells failed to protect CFU-GM from Ara-C-induced cytotoxicity.