RESUMEN
Although some information is available for specific subsets of miRNAs and several factors have been shown to bind oligonucleotides (ONs), no general transport mechanism for these molecules has been identified to date. In this work, we demonstrate that the nuclear transport of ONs, siRNAs, and miRNAs responds to cellular stress. Furthermore, we have identified a stress-induced response complex (SIRC), which includes Ago-1 and Ago-2 in addition to the transcription and splicing regulators YB1, CTCF, FUS, Smad1, Smad3, and Smad4. The SIRC transports endogenous miRNAs, siRNAs, and ONs to the nucleus. We show that cellular stress can significantly increase ON- or siRNA-directed splicing switch events and endogenous miRNA targeting of nuclear RNAs.
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Núcleo Celular/metabolismo , MicroARNs/metabolismo , Oligonucleótidos/metabolismo , ARN Interferente Pequeño/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Transcripción Genética/fisiologíaRESUMEN
Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.
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Citoplasma/metabolismo , Silenciador del Gen , Oligonucleótidos Antisentido , Proteínas Argonautas/metabolismo , Línea Celular , Citoplasma/química , Técnicas de Transferencia de Gen , Oligonucleótidos Antisentido/análisis , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , TransfecciónAsunto(s)
Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/secundario , Antineoplásicos Hormonales/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Mitotano/administración & dosificación , Neoplasias de la Corteza Suprarrenal/cirugía , Adrenalectomía , Esquema de Medicación , Femenino , Humanos , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Factores de Tiempo , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.
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Antineoplásicos/uso terapéutico , ADN sin Sentido/uso terapéutico , ADN de Cadena Simple/uso terapéutico , Silenciador del Gen/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Región de Flanqueo 5'/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN sin Sentido/administración & dosificación , ADN sin Sentido/farmacocinética , ADN sin Sentido/farmacología , ADN de Cadena Simple/administración & dosificación , ADN de Cadena Simple/farmacocinética , ADN de Cadena Simple/farmacología , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Liposomas , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Neoplasias/sangre , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacocinética , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/uso terapéutico , Vehículos Farmacéuticos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: This multicenter phase II trial evaluated the efficacy and safety of trabectedin in metastatic castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: Two schedules were evaluated in three cohorts: weekly as 3-h i.v. infusion at 0.58 mg/m(2) for 3 out of 4 weeks (Cohort A, n = 33), and every 3 weeks (q3wk) as 24-h infusion at 1.5 mg/m(2) (Cohort B1, n = 5) and 1.2 mg/m(2) (Cohort B2, n = 20). The primary end point was prostate-specific antigen (PSA) response; secondary end points included safety, tolerability and time to progression (TTP). RESULTS: Trabectedin resulted in PSA declines ≥ 50% in 12.5% (Cohort A) and 10.5% (Cohort B2) of patients. Among men pretreated with taxane-based chemotherapy, PSA response was 13.6% (Cohort A) and 15.4% (Cohort B2). PSA responses lasted 4.1-8.6 months, and median TTP was 1.5 months (Cohort A) and 1.9 months (Cohort B2). The dose of 1.5 mg/m(2) (approved for soft tissue sarcoma) given as 24-h infusion q3wk was not tolerable in these patients. At 1.2 mg/m(2) q3wk and 0.58 mg/m(2) weekly, the most common adverse events were nausea, fatigue and transient neutropenia and transaminase increase. CONCLUSIONS: Two different trabectedin schedules showed modest activity in metastatic CRPC. Further studies may require identification of predictive factors of response in prostate cancer.
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Dioxoles/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Sarcoma/tratamiento farmacológico , Tetrahidroisoquinolinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Alquilantes/efectos adversos , Antineoplásicos Alquilantes/uso terapéutico , Estudios de Cohortes , Dioxoles/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Orquiectomía , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Sarcoma/patología , Sarcoma/cirugía , Tetrahidroisoquinolinas/efectos adversos , Trabectedina , Insuficiencia del Tratamiento , Resultado del TratamientoRESUMEN
Antisense oligodeoxyribonucleotides have been used for decades to achieve sequence-specific silencing of gene expression. However, all early generation oligonucleotides (e.g., those with no other modifications than the phosphorothioate backbone) are inactive in vitro unless administered using a delivery vehicle. These delivery vehicles are usually lipidic but can also be polyamines or some other particulate reagent. We have found that by employing locked nucleic acid (LNA) phosphorothioate gap-mer nucleic acids of 16 mer or less in length, and by carefully controlling the plating conditions of the target cells and duration of the experiment, sequence-specific gene silencing can be achieved at low micromolar concentrations in vitro in the absence of any delivery vehicle. This process of naked oligonucleotide delivery to achieve gene silencing in vivo, which we have termed gymnosis, has been observed in many both adherent and nonadherent cell lines against several different targets genes.
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Técnicas de Silenciamiento del Gen/métodos , Silenciador del Gen , Oligodesoxirribonucleótidos Antisentido/genética , Secuencia de Bases , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Oligonucleótidos/genética , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/métodosRESUMEN
For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
Asunto(s)
Silenciador del Gen , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos/administración & dosificación , Animales , Línea Celular Tumoral , Humanos , Indicadores y Reactivos , Ratones , Oligonucleótidos/análisis , Oligonucleótidos Antisentido/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , TransfecciónRESUMEN
PURPOSE: We examined the effects of G3139 on the interaction of heparin-binding proteins [e.g., fibroblast growth factor 2 (FGF2) and collagen I] with endothelial cells. G3139 is an 18-mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global phase III trial in advanced melanoma (GM301) has evaluated G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood because it is unlikely that Bcl-2 silencing is the sole mechanism for chemosensitization in melanoma cells. EXPERIMENTAL DESIGN: The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high-affinity cell surface receptors and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and human umbilical vascular endothelial cells were determined. RESULTS: G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in three-dimensional collagen gels, increases the mitogenesis of human umbilical vascular endothelial cells similarly, and induces vessel sprouts in the rat aortic ring model. CONCLUSIONS: G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with lactate dehydrogenase observed clinically.
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Células Endoteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Oligonucleótidos Antisentido/farmacología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Tionucleótidos/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Becaplermina , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Humanos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-sis , Ratas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/efectos de los fármacos , Tionucleótidos/genética , Tionucleótidos/metabolismoRESUMEN
Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.
Asunto(s)
Células Endoteliales/metabolismo , Fibrinolíticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Enfermedad Veno-Oclusiva Hepática/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Polidesoxirribonucleótidos/farmacología , Animales , Trasplante de Médula Ósea/efectos adversos , Bovinos , Línea Celular Transformada , Córnea/metabolismo , Córnea/patología , Células Endoteliales/patología , Fibrinolíticos/uso terapéutico , Enfermedad Veno-Oclusiva Hepática/tratamiento farmacológico , Enfermedad Veno-Oclusiva Hepática/etiología , Enfermedad Veno-Oclusiva Hepática/patología , Humanos , Insuficiencia Multiorgánica/tratamiento farmacológico , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/metabolismo , Insuficiencia Multiorgánica/patología , Polidesoxirribonucleótidos/uso terapéutico , Unión Proteica/efectos de los fármacos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
VDAC channels are ancient, highly-conserved voltage-gated channels in the mitochondrial outer membrane. They are the pathways by which metabolites travel between the cytosol and mitochondria. They are involved in the apoptotic process and probably other functions as well. The lack of specific inhibitors has hampered research in the past but now phosphorothioate oligonucleotides can serve this function. These molecules were generated to be stable in the cytosol of cells but, unlike the oligonucleotides with the physiological phosphodiester linkage, these have the ability to bind to and block VDAC channels. They are potent, specific, and available commercially. At 1 microM concentration they block VDAC channels in mitochondria but do not affect the respiration complexes, the adenine nucleotide translocator or the ATP synthase.
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Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Oligonucleótidos Fosforotioatos/farmacología , Canales Aniónicos Dependientes del Voltaje/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Mitocondriales/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Dacarbazina/uso terapéutico , Melanoma/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Tionucleótidos/uso terapéutico , Aprobación de Drogas , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Estados Unidos , United States Food and Drug AdministrationAsunto(s)
Tionucleótidos/uso terapéutico , Ensayos Clínicos como Asunto , Determinación de Punto Final , Humanos , L-Lactato Deshidrogenasa/metabolismo , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pronóstico , Análisis de Supervivencia , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bcl-2-independent apoptosis, perhaps partly via direct interaction with VDAC and reduction of metabolite flow across the mitochondrial outer membrane. Here, we analyzed the interactions at the molecular level. Ten micromolar G3139 induces rapid flickering of the VDAC conductance and, occasionally, a complete conductance drop. These phenomena occur only when VDAC is in the "open" conformation and therefore are consistent with pore blockage rather than VDAC closure. Blockage occurs preferentially from one side of the VDAC channel. It depends linearly on the [G3139] and is voltage-dependent with an effective valence of -3. The kinetics indicate at least a partial entry of G3139 into VDAC, forming an unstable bound state, which is responsible for the rapid flickering (approximately 0.1 ms). Subsequently, a long-lived blocked state is formed. An 8-mer phosphorothioate, polydeoxythymidine, induces partial blockage of VDAC and a change in selectivity from favoring anions to favoring cations. Thus, the oligonucleotide is close to the ion stream. The phosphodiester congener of G3139 is ineffective at the concentrations used, excluding a general polyanion effect. This shows the importance of sulfur atoms. The results are consistent with a binding-induced blockage rather than a permeation block.
Asunto(s)
Oligonucleótidos Antisentido/química , Compuestos Organotiofosforados/química , Poli T/química , Tionucleótidos/química , Canales Aniónicos Dependientes del Voltaje/antagonistas & inhibidores , Canales Aniónicos Dependientes del Voltaje/química , Animales , Activación del Canal Iónico , Cinética , Membranas Artificiales , Mitocondrias Hepáticas/química , Conformación Proteica , RatasRESUMEN
In order to overcome the problems of enzymatic degradation and short plasma half life, which can limit the delivery of antisense oligonucleotides, and the potential immuno-stimulatory effects of CpG motifs, we utilized a polyethylene glycol (PEG) technology that employed various releasable linkers (rPEG). 5'-20 kDa-PEGylation of an anti-Bcl-2 5'-aminoalkyl-oligonucleotide with the same sequence as G3139 (Compound 1) did not alter its binding to the heparin-binding protein bFGF, nor the release of cytochrome c from isolated mitochondria treated with the conjugates. However, in 518A2 melanoma cells in vitro, PEGylation resulted in greatly diminished cellular uptake. In striking contrast, PEGylation of 1 resulted in dramatically improved pharmacokinetic profiles in vivo, with a prolonged half-life (t1/2), increased plasma concentration, and increased area under the plasma concentration-time curve (AUC). In an in vivo melanoma 518A2 xenograft mouse model, treatment with either 5'-20 kDa-PEG-1 or 1 demonstrated similar tumor growth inhibition. Furthermore, in an in vitro mouse splenocyte culture system, attachment of a PEG moiety to 1 through releasable linkers abolished the immunostimulatory response that was observed for G3139. Our results demonstrate the potential of the in vivo use of PEGylated oligonucleotides, and point out the profound differences between in vitro and in vivo models of oligonucleotide activity.
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Antineoplásicos/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Polietilenglicoles/farmacocinética , Tionucleótidos/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Polietilenglicoles/administración & dosificación , Ratas , Ratas Sprague-Dawley , Tionucleótidos/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosAsunto(s)
Antineoplásicos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Hipercolesterolemia/tratamiento farmacológico , Cetoconazol/efectos adversos , Lovastatina/efectos adversos , Neoplasias de la Próstata/tratamiento farmacológico , Rabdomiólisis/inducido químicamente , Anciano de 80 o más Años , Interacciones Farmacológicas , Humanos , Hipercolesterolemia/complicaciones , Masculino , Neoplasias de la Próstata/complicaciones , Resultado del TratamientoRESUMEN
G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K(i) between 0.2 and 0.5 microM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 microM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 microM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.
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Adenosina Difosfato/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Tionucleótidos/farmacología , Animales , Línea Celular Tumoral , Citocromos c/metabolismo , Humanos , Técnicas In Vitro , Activación del Canal Iónico , Liposomas/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Permeabilidad , Fosfolípidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Canales Aniónicos Dependientes del Voltaje/fisiologíaRESUMEN
PURPOSE: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. EXPERIMENTAL DESIGN: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. RESULTS: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors. CONCLUSIONS: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.
Asunto(s)
Melanoma/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Apoptosis/genética , Secuencia de Bases , Citocromos c/metabolismo , ADN sin Sentido/genética , Regulación hacia Abajo , Silenciador del Gen , Humanos , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Ratones , Ratones Endogámicos ICR , Ratones SCID , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Tionucleótidos/genética , Tionucleótidos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
G3139, an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in 518A2 and other melanoma cells. G3139-mediated apoptosis appears to be independent of its ability to down-regulate the expression of Bcl-2 protein, because the release of mitochondrial cytochrome c precedes in time the down-regulation of Bcl-2 protein expression. In this study, we demonstrate the ability of G3139 and other phosphorothioate oligonucleotides to bind directly to mitochondria isolated from 518A2 cells. Furthermore, we show that this interaction leads to the release of cytochrome c in the absence of a mitochondrial membrane permeability transition. Our data further demonstrate that there is an interaction between G3139 and VDAC, a protein that can facilitate the physiologic exchange of ATP and ADP across the outer mitochondrial membrane. Evidence from the electrophysiologic evaluation of VDAC channels reconstituted into phospholipid membranes demonstrates that G3139 is capable of producing greatly diminished channel conductance, indicating a closed state of the VDAC. This effect is oligomer length-dependent, and the ability of phosphorothioate homopolymers of thymidine of variable lengths to cause the release of cytochrome c from isolated mitochondria of 518A2 melanoma cells can be correlated with their ability to interact with VDAC. Because it has been suggested that the closure of VDAC leads to the opening of another outer mitochondrial membrane channel through which cytochrome c can transit, thus initiating apoptosis, it appears that VDAC may be an important pharmacologic target of G3139.
Asunto(s)
Melanoma/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Tionucleótidos/farmacología , Canales Aniónicos Dependientes del Voltaje/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Citocromos c/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Fosfatos/químicaRESUMEN
Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Tionucleótidos/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Linfocitos B/efectos de los fármacos , Carcinoma/tratamiento farmacológico , Carcinoma/inmunología , Línea Celular Tumoral , Citosina/química , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Activación de Linfocitos , Metilación , Ratones , Ratones Desnudos , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/química , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tionucleótidos/administración & dosificación , Tionucleótidos/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Veno-occlusive disease of the liver (VOD) remains a troubling and potentially fatal complication of high-dose chemotherapy and hematopoietic stem cell transplantation conditioning regimens. No effective therapy has been available for these patients to date, and the best supportive care measures remain woefully inadequate. Defibrotide (DF) (Gentium, S.p.A., Como, Italy), a polydisperse mixture of all the single-stranded phosphodiester oligodeoxyribonucleotides that can be obtained from the controlled depolymerization of porcine intestinal mucosal genomic DNA, seems to offer a safe and effective treatment for some patients suffering from severe VOD, a condition for which no accepted standard therapy currently exists. Early clinical studies evaluating the efficacy of DF for the treatment of severe VOD in patients undergoing hematopoietic stem cell transplantation have been very encouraging. Approximately 45% of the patients treated in multiple initial phase II clinical trials achieved a complete response at day +100, demonstrating normalization of serum bilirubin and resolution of the clinical syndrome. However, although multi-institutional, these represented single arm studies. A large, FDA-approved, pivotal, prospective, multi-institutional, global phase III trial of DF vs. historical controls (best available therapy) commenced in the first quarter of 2006 and should provide further validation of DF's efficacy. The drug seems to have few significant side effects, and almost all test subjects who have received this treatment have tolerated it well. Although the mechanism of action remains unclear, the drug exerts minimal systemic anticoagulant effects yet appears to induce numerous antithrombotic and profibrinolytic effects both in vitro and in vivo. It may function as an adenosine receptor agonist and causes increased concentrations of endogenous prostaglandins, which modulate thrombomodulin, platelets, and fibrinolysis. It also appears to block lipopolysaccharide (LPS)-induced tissue factor (TF) expression. However, despite the fact the DF is composed of oligonucleotides, its mechanism of action, which at the present time is unclear, is not related to Watson-Crick base pair-dependent downregulation of gene expression but is rather likely a result of its polyanionic nature.