RESUMEN
Chemotherapy resistance is the major cause of treatment failure in osteosarcoma, the most common primary bone malignancy, and sensitizing therapeutic strategy is required to improve the clinical outcome. In this study, we discovered that navitoclax, a selective inhibitor of Bcl-2/Bcl-xL, effectively combats chemoresistance in osteosarcoma. Our research revealed that Bcl-2, but not Bcl-xL, is upregulated in osteosarcoma cells that are resistant to doxorubicin. However, venetoclax, a specific inhibitor of Bcl-2, did not exhibit activity against doxorubicin-resistant cells. Further analysis showed that depleting either Bcl-2 or Bcl-xL alone was insufficient to overcome doxorubicin resistance. Only by depleting both Bcl-2 and Bcl-xL significantly reduce the viability of doxorubicin-resistant cells. Similarly, navitoclax not only decreased the viability of doxorubicin-resistant cells but also acted synergistically with doxorubicin in cells sensitive to the drug. To confirm the ability of navitoclax to overcome doxorubicin resistance, we conducted experiments using multiple mouse models of osteosarcoma, both doxorubicin-sensitive and doxorubicin-resistant. The results provided confirmation that navitoclax is effective in overcoming doxorubicin resistance. Our findings demonstrate that simultaneous inhibition of Bcl-2 and Bcl-xL could serve as a novel strategy to sensitize chemoresistant osteosarcoma cells. Moreover, our study presents preclinical evidence supporting the potential of a navitoclax and doxorubicin combination therapy for the treatment of osteosarcoma, paving the way for future clinical investigations.
Asunto(s)
Compuestos de Anilina , Neoplasias Óseas , Osteosarcoma , Sulfonamidas , Animales , Ratones , Proteína bcl-X/farmacología , Proteína bcl-X/uso terapéutico , Apoptosis , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Resistencia a AntineoplásicosRESUMEN
Axonal degeneration of retinal ganglion cells (RGCs) causes blindness in glaucoma. Currently, there are no therapies that target axons to prevent them from degenerating. Activation of the BAX protein has been shown to be the determining step in the intrinsic apoptotic pathway that causes RGCs to die in glaucoma. A putative role for BAX in axonal degeneration is less well elucidated. BCLXL (BCL2L1) is the primary antagonist of BAX in RGCs. We developed a mCherry-BCLXL fusion protein, which prevented BAX recruitment and activation to the mitochondria in tissue culture cells exposed to staurosporine. This fusion protein was then packaged into adeno-associated virus serotype 2, which was used to transduce RGCs after intravitreal injection and force its overexpression. Transduced RGCs express mCherry-BCLXL throughout their somas and axons along the entire optic tract. In a model of acute optic nerve crush, the transgene prevented the recruitment of a GFP-BAX fusion protein to mitochondria and provided long-term somal protection up to 12 weeks post injury. To test the efficacy in glaucoma, DBA/2J mice were transduced at 5 months of age, just prior to the time they begin to exhibit ocular hypertension. Gene therapy with mCherry-BCLXL did not affect the longitudinal history of intraocular pressure elevation compared to naive mice but did robustly attenuate both RGC soma pathology and axonal degeneration in the optic nerve at both 10.5 and 12 months of age. BCLXL gene therapy is a promising candidate for glaucoma therapy.
Asunto(s)
Terapia Genética , Glaucoma/terapia , Neuronas/patología , Proteína bcl-X/genética , Proteína bcl-X/uso terapéutico , Envejecimiento/patología , Animales , Dependovirus , Modelos Animales de Enfermedad , Glaucoma/complicaciones , Glaucoma/fisiopatología , Proteínas Fluorescentes Verdes/metabolismo , Presión Intraocular , Ratones Endogámicos DBA , Mitocondrias/metabolismo , Compresión Nerviosa , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/patología , Nervio Óptico/metabolismo , Nervio Óptico/patología , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Ganglionares de la Retina/metabolismoRESUMEN
Most cells express a variety of both anti-apoptotic and pro-apoptotic Bcl-2 proteins and the interaction within this family dictates whether a cell survives or dies. The dysregulation of the anti-anti-apoptotic Bcl-2 family members is one of the defining features of cancer cells in comparison to normal cells, and significantly contributes to the resistance of cancer cells to current treatment modalities. This anti-apoptotic subfamily of proteins is now a major target in the development of new methods to improve treatment outcomes for cancer patients. Several drugs directed at inhibiting Bcl-2 and related anti-apoptotic proteins have been developed with some showing considerable promise in the clinic. This Review presents the current knowledge of the role of the anti-apoptotic Bcl-2 family in cancer cells, as well as current and future perspectives on targeting this subfamily of proteins for therapeutic intervention in human malignancies. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".
Asunto(s)
Proteínas Reguladoras de la Apoptosis/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Reguladoras de la Apoptosis/clasificación , Proteínas Reguladoras de la Apoptosis/metabolismo , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/clasificación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/uso terapéutico , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismo , Proteína bcl-X/uso terapéuticoRESUMEN
We previously reported that the protein transduction domain fused FNK (PTD-FNK) protein, which was derived from anti-apoptotic Bcl-xL protein and thereby gained higher anti-cell death activity, has a strong neuroprotective effect on rat focal brain ischemia models. The aim of this study was to investigate the effect of PTD-FNK protein and hypothermia combined therapy on cerebral infarction. Male SD rats were subjected to 120min middle cerebral artery occlusion (MCAO) with intraluminal thread. Rats were divided into 4 groups: 1) 37°C vehicle administration (37V); 2) 37°C PTD-FNK administration (37F); 3) 35°C vehicle administration (35V); and 4) 35°C PTD-FNK administration (35F). PTD-FNK protein was intravenously administered 60min after the induction of MCAO. Hypothermia (35°C) was applied during 120min MCAO. Rats were sacrificed 24h later; infarct volumes were measured, and Bax, Bcl-2, TUNEL and caspase-12 immunostaining was evaluated. There was significant infarct volume reduction in 37F, 35V, and 35F groups compared to 37V. There was also a significant difference between 37F and 35F. This suggests that hypothermia enhanced the effect of PTD-FNK. Similar results were found in neurological symptoms. Caspase-12 and TUNEL staining showed a significant difference between 37F and 35F; however, Bax and Bcl-2 staining failed to show a difference. In this study we showed an additive protective effect of hypothermia on PTD-FNK treatment, and immunohistological results showed that the protective mechanisms might involve the inhibition of apoptotic pathways through caspase-12, but not through Bcl-2.
Asunto(s)
Infarto Encefálico/terapia , Hipotermia Inducida/métodos , Ataque Isquémico Transitorio/terapia , Fármacos Neuroprotectores/farmacología , Proteínas Serina-Treonina Quinasas/uso terapéutico , Proteína bcl-X/uso terapéutico , Animales , Apoptosis/genética , Infarto Encefálico/metabolismo , Infarto Encefálico/patología , Caspasa 12/genética , Caspasa 12/uso terapéutico , Modelos Animales de Enfermedad , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patología , Masculino , Fármacos Neuroprotectores/síntesis química , Proteínas Serina-Treonina Quinasas/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Supresoras de Tumor , Proteína bcl-X/genéticaRESUMEN
There is an obvious need to develop pharmacological strategies to protect the heart in patients suffering from acute myocardial infarction. Apoptosis was evidenced as a main contributor of myocardial ischemia-reperfusion (IR) injury. Our cardioprotective strategy was based on the use of four cell penetrating peptides (CPP: Tat, (RXR)4, Bpep and Pip2b) which were conjugated to the BH4-peptide, derived from the BH4 domain of the Bcl-xL anti-apoptotic protein. These CPP-BH4 conjugates were able to reduce staurosporine-induced apoptosis in primary cardiomyocytes in vitro. Although Pip2b-BH4 was more efficient in terms of cellular uptake, it was as efficient as Tat-BH4 for its anti-apoptotic activity. As required for potential therapeutic application their cardioprotective effects were evaluated in an in vivo mouse model of myocardial IR injury. Our results clearly show that a single low dose (1 mg/kg) injection of Tat-BH4 and Pip2b-BH4 administered intravenously 5 min before reperfusion was able to drastically reduce infarct size (~47%) and to inhibit apoptosis (~60%) in the left ventricle of treated mice. Importantly, these effects are not observed following the injection of CPP alone or scrambled version of BH4. This study evidences that the Pip2b CPP, designed for oligonucleotides translocation, as well as the widely used natural Tat CPP exhibit similar efficacy in vivo to deliver BH4 anti-apoptotic peptide to the reperfused myocardium and may thus become useful therapeutic tools to treat acute myocardial infarction in the clinical setting.
Asunto(s)
Apoptosis/efectos de los fármacos , Daño por Reperfusión Miocárdica/prevención & control , Péptidos/química , Péptidos/uso terapéutico , Proteína bcl-X/química , Proteína bcl-X/uso terapéutico , Secuencia de Aminoácidos , Animales , Línea Celular , Péptidos de Penetración Celular/química , Células Cultivadas , Corazón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patologíaRESUMEN
OBJECTIVE: To indentify the relation between hepatic cells apoptosis and the lesion of liver tissue in acute toxic lethal hepatitis. METHODS: 60 Wistar mice were randomly divided into normal control, model group and treatment group. Normal control and model group were pretreated by portal vein injection of normal saline, the treatment group was pretreated by portal vein injection of BCL-X1 adenoviruses. The mice of model group and treatment group were received an injection of D-galn and LPS to establish fulminant hepatic failure models 7 days after pretrement. To observe BCL-X1 expression, serum ALT, AST, hepatocyte apoptosis rate, and mortality rate of the three groups. RESULTS: The BCL-X1 expression was higher in treatment group than in model group; 6 hours after fulminant hepatic failure models were established,the serum ALT, AST level of treatment group was lower than model group;The hepatocyte apoptosis rate of treatment group was lower than model group. The death rate of treatment group was lower than model group. CONCLUSION: In fulminant mice hepatic failure models, the hepatocyte apoptosis rate has a positive correlation with death rate, the overexpression of BCL-X1 can decrease the hepatocyte apoptosis rate and the death rate.
Asunto(s)
Adenoviridae/genética , Expresión Génica , Fallo Hepático Agudo/genética , Fallo Hepático Agudo/prevención & control , Proteína bcl-X/genética , Adenoviridae/metabolismo , Animales , Apoptosis , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Hígado/citología , Hígado/metabolismo , Fallo Hepático Agudo/fisiopatología , Fallo Hepático Agudo/terapia , Ratas , Ratas Wistar , Proteína bcl-X/metabolismo , Proteína bcl-X/uso terapéuticoRESUMEN
Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.
Asunto(s)
Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Células-Madre Neurales/trasplante , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/terapia , Transducción Genética/métodos , Proteína bcl-X/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/cirugía , Supervivencia Celular/genética , Citoprotección/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Recuperación de la Función/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Tiempo , Proteína bcl-X/uso terapéutico , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/uso terapéuticoRESUMEN
We investigated the synergism between shRNAs against Bcl-xL and doxorubicin (DOX) using aptamer-conjugated polyplexes (APs) in combination cancer therapy. Synergistic and selective cancer cell death was achieved by AP-mediated co-delivery of very small amounts of DOX and Bcl-xL-specific shRNA, which simultaneously activated an intrinsic apoptotic pathway. A branched polyethyleneimine (PEI) was grafted to polyethylene glycol (PEI-PEG) to serve as a vehicle for shRNA delivery, and its surface was further conjugated with an anti-PSMA aptamer (APT) for the selective delivery of APs to prostate cancer cells that express prostate-specific membrane antigens (PSMA) on their cell surface. The APs were finally obtained after intercalation of DOX to form shRNA/PEI-PEG-APT/DOX conjugates. Cell viability assays and FACS analysis of GFP expression against PC3 (PSMA deficient) and LNCaP (PSMA overexpressed) cells demonstrated that the synthesized APs inhibited the growth of PSMA-abundant prostate cancer cells with strong cell selectivity. Consequently, IC(50) values of APs loaded with both DOX and shRNA were approximately 17-fold less than those for the simple mixture of shRNA plus drug (shRNA/Lipofectamine + DOX). These results suggest that AP-mediated co-delivery of an anti-cancer drug and shRNA against Bcl-xL may widen the therapeutic window and allow for the selective destruction of cancer cells.
Asunto(s)
Antibióticos Antineoplásicos , Aptámeros de Nucleótidos , Muerte Celular/efectos de los fármacos , Doxorrubicina , Neoplasias de la Próstata/tratamiento farmacológico , ARN Interferente Pequeño , Proteína bcl-X , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/uso terapéutico , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Humanos , Masculino , Ensayo de Materiales , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Polietileneimina/química , Polietileneimina/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/uso terapéuticoRESUMEN
Direct intra-articular injection of mesenchymal stem cells (MSCs) has been proposed as a potential cell therapy for cartilage defects. This cell therapy relies on the survival of the implanted MSCs. However, the arduous local environment may limit cell viability after implantation, which would restrict the cells' regenerative capacity. Thus, it is necessary to reinforce the implanted cells against the unfavourable microenvironment in order to improve the efficacy of cell therapy. We examined whether the transduction of an anti-apoptotic protein, Bcl-xL, into MSCs could prevent cell death and improve the implantation efficiency of MSCs in a rabbit model. Our current findings demonstrate that the group treated with Bcl-xL-engineered MSCs could improve cartilage healing both morphologically and histologically when compared with the controls. These results suggest that intra-articular injection of Bcl-xL-engineered MSCs is a potential non-invasive therapeutic method for effectively treating cartilage defects of the knee.
Asunto(s)
Cartílago Articular/patología , Terapia Genética , Miembro Posterior/patología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Proteína bcl-X/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Cartílago Articular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Miembro Posterior/efectos de los fármacos , Humanos , Etiquetado Corte-Fin in Situ , Inyecciones Intraarticulares , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Conejos , Transfección , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Cuerpo Estriado/fisiopatología , Productos del Gen tat/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Células Madre/fisiología , Proteína bcl-X/uso terapéutico , Animales , Isquemia Encefálica/fisiopatología , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Aprendizaje , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Actividad Motora , Neurogénesis/fisiología , Neuropéptidos/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Percepción EspacialRESUMEN
A number of gene therapy approaches have been developed for protecting neurons from necrotic neurological insults. Such therapies are limited by the need for transcription and translation of the protective protein, delaying therapeutic impact. As an alternative, we explore the neuroprotective potential of protein therapy, using a fusion protein comprised of the death-suppressing BH4 domain of the Bcl-xL protein and the protein transduction domain of the human immunodeficiency virus Tat protein. This fusion protein decreased neurotoxicity caused by the excitotoxins glutamate and kainic acid in primary hippocampal cultures, and decreased hippocampal damage in vivo in an excitotoxic seizure model.
Asunto(s)
Apoptosis/fisiología , Productos del Gen tat/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Proteína bcl-X/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Embrión de Mamíferos , Productos del Gen tat/biosíntesis , Ácido Glutámico/toxicidad , Hipocampo/citología , Ácido Kaínico/toxicidad , Masculino , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteína bcl-X/metabolismoRESUMEN
Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immune-privileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium.
Asunto(s)
Apoptosis/fisiología , Trasplante de Córnea/patología , Endotelio Corneal/patología , Terapia Genética/métodos , Rechazo de Injerto/patología , Proteína bcl-X/uso terapéutico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
In multiple sclerosis (MS), post-mortem studies of human brain tissue as well as data from animal models have shown that apoptosis of neurons occurs to a significant extent during this disease. As neurodegeneration in MS correlates with permanent neurological deficits in patients, understanding the mechanisms would be an important pre-condition for designing appropriate neuroprotective therapies. Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis often affects the optic nerve and leads to consecutive apoptosis of retinal ganglion cells (RGCs), the neurons that form its axons. In this study, we fused Bcl-XL to the protein transduction domain of the HIV-transactivator of transcription. Thereby, this anti-apoptotic member of the Bcl-2 family was delivered into RGCs of rats with electrophysiologically diagnosed optic neuritis. Transduction of Bcl-XL in our study led to significant rescue of RGCs indicating the relevance of this pathway for neuronal survival under autoimmune inflammatory conditions.