RESUMEN
Glioblastoma (GB) is the most aggressive and common form of primary brain tumor characterized by fast proliferation, high invasion and resistance to current standard treatment. The average survival rate post-diagnosis is 14.6 months, despite the aggressive standard post-surgery radiotherapy concomitant with chemotherapy with temozolomide (TMZ). Currently, efforts are being endowed to develop new and more efficient therapeutic approaches capable to overcome chemoresistance, inhibit tumor progression and improve overall patient survival rate. Abnormal microRNA (miRNA) expression has been correlated with chemoresistance, proliferation and resistance to apoptosis, which result from their master regulatory role of gene expression. Altered cell metabolism, favoring glycolysis, was identified as an emerging cancer hallmark and has been described in GB, thus offering a new target for innovative GB therapies. In this work, we hypothesized that a gene therapy-based strategy consisting of the overexpression of a miRNA downregulated in GB and predicted to target crucial metabolic enzymes might promote a shift of GB cell metabolism, decreasing the glycolytic dependence of tumor cells and contributing to their sensitization to chemotherapy with TMZ. The increase of miR-200c levels in DBTRG cells resulted in downregulation of messenger RNA of enzymes involved in bioenergetics pathways and impaired cell metabolism and mobility. In addition, miR-200c overexpression prior to DBTRG cell exposure to TMZ resulted in cell cycle arrest. Overall, our results show that miR-200c overexpression could offer a way to overcome chemoresistance developed by GB cells in response to current standard chemotherapy, providing an improvement to current GB standard treatment, with benefit for patient outcome.
Asunto(s)
Resistencia a Antineoplásicos/genética , Metabolismo Energético , Glioblastoma/genética , Glioblastoma/metabolismo , MicroARNs/genética , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Interferencia de ARN , ARN MensajeroRESUMEN
Despite the intense global efforts towards an effective treatment of glioblastoma (GB), current therapeutic options are unsatisfactory with a median survival time of 12-15 months after diagnosis, which has not improved significantly over more than a decade. The high tumoral heterogeneity confers resistance to therapies, which has hindered a successful clinical outcome, GB remaining among the deadliest cancers. A hallmark of GB is its high recurrence rate, which has been attributed to the presence of a small subpopulation of tumor cells called GB stem-like cells (GSC). In the present work, the efficacy of a multimodal strategy combining microRNA (miRNA) modulation with new generation multitargeted tyrosine kinase inhibitors (imatinib and axitinib) was investigated aiming at tackling this subpopulation of GB cells. MiR-128 and miR-302a were selected as attractive therapeutic candidates on the basis of previous findings reporting that reestablishment of their decreased expression levels in GSC resulted in cell differentiation, which could represent a possible strategy to sensitize GSC to chemotherapy. Our results show that overexpression of miR-128 or miR-302a induced GSC differentiation, which enhanced senescence mediated by axitinib treatment, thus further impairing GSC proliferation. We also provided evidence for the capacity of GSC to efficiently internalize functionalized stable nucleic acid lipid particles, previously developed and successfully applied in our laboratory to target GB. Taken together, our findings will be important in the future design of a GB-targeted multimodal miRNA-based gene therapy, combining overexpression of miR-128 or miR-302a with axitinib treatment, endowed with the ability to overcome drug resistance.
Asunto(s)
Axitinib/uso terapéutico , Diferenciación Celular , Glioblastoma/tratamiento farmacológico , MicroARNs/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Axitinib/farmacología , Línea Celular Tumoral , Terapia Combinada , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/fisiopatología , Humanos , Mesilato de Imatinib/farmacología , MicroARNs/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Regulación hacia ArribaRESUMEN
Glioblastoma (GB) is the most frequent and malignant type of brain tumor, for which no effective therapy exists. The high proliferative and invasive nature of GB, as well as its acquired resistance to chemotherapy, makes this type of cancer extremely lethal shortly after diagnosis. Long non-protein coding RNAs (lncRNA) are a class of regulatory RNAs whose levels can be dysregulated in the context of diseases, unbalancing several physiological processes. The lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA-MVIH), overexpressed in several cancers, was described to co-precipitate with phosphoglycerate kinase 1 (PGK1), preventing secretion of this enzyme to the extracellular environment and promoting cell migration and invasion. We hypothesized that, by silencing the expression of lncRNA-MVIH, the secretion of PGK1 would increase, reducing GB cell migration and invasion capabilities. We observed that lncRNA-MVIH silencing in human GB cells significantly decreased glycolysis, cell growth, migration, and invasion and sensitized GB cells to cediranib. However, no increase in extracellular PGK1 was observed as a consequence of lncRNA-MVIH silencing, and therefore, we investigated the possibility of a mechanism of miRNA sponge of lncRNA-MVIH being in place. We found that the levels of miR-302a loaded onto RISC increased in GB cells after lncRNA-MVIH silencing, with the consequent downregulation of several miR-302a molecular targets. Our findings suggest a new mechanism of action of lncRNA-MVIH as a sponge of miR-302a. We suggest that lncRNA-MVIH knockdown may be a promising strategy to address GB invasiveness and chemoresistance, holding potential towards its future application in a clinical context.
Asunto(s)
Glioblastoma/genética , MicroARNs/genética , Fosfoglicerato Quinasa/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/patología , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patologíaRESUMEN
Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.
Asunto(s)
Glioblastoma , Estearoil-CoA Desaturasa , Humanos , Estearoil-CoA Desaturasa/metabolismo , Fosfolípidos , Glioblastoma/genética , Autofagia/genética , Supervivencia Celular/genéticaRESUMEN
A great deal of evidence revealing that lipid metabolism is drastically altered during tumorigenesis has been accumulated. In this work, glucosylceramide synthase (GCS) was targeted, using RNA interference technology (siRNAs), in U87 and DBTRG human glioblastoma (GBM) cells, as in both cell types GCS showed to be overexpressed with respect to normal human astrocytes. The efficacy of a combined therapy to tackle GBM, allying GCS silencing to the new generation chemotherapeutics sunitinib and axitinib, or to the alkylating drugs etoposide and temozolomide, is evaluated here for the first time. With this purpose, studies addressing GBM cell viability and proliferation, cell cycle and apoptosis were performed, which revealed that combination of GCS silencing with axitinib treatment represents a promising therapeutic approach. The reduction of cell viability induced by this combined therapy is proposed to be mediated by excessive production of reactive oxygen species. This work, identifying GCS as a key molecular target to increase GBM susceptibility to a new generation chemotherapeutic, opens windows to the development of innovative strategies to halt GBM recurrence after surgical resection.
Asunto(s)
Axitinib/farmacología , Glioblastoma/genética , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/genética , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/fisiopatología , Glucosiltransferasas/metabolismo , Humanos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Glioblastoma (GB) is the most aggressive and common form of primary brain tumor, characterized by fast proliferation, high invasion, and resistance to current standard treatment. The average survival rate post-diagnosis is only of 14.6 months, despite the aggressive standard post-surgery treatment approaches of radiotherapy concomitant with chemotherapy with temozolomide. Altered cell metabolism has been identified as an emerging cancer hallmark, including in GB, thus offering a new target for cancer therapies. On the other hand, abnormal expression levels of miRNAs, key regulators of multiple molecular pathways, have been correlated with pathological manifestations of cancer, such as chemoresistance, proliferation, and resistance to apoptosis. In this work, we hypothesized that gene therapy based on modulation of a miRNA with aberrant expression in GB and predicted to target crucial metabolic enzymes might impair tumor cell metabolism. We found that the increase of miR-144 levels, shown to be downregulated in U87 and DBTRG human GB cell lines, as well as in GB tumor samples, promoted the downregulation of mRNA of enzymes involved in bioenergetic pathways, with consequent alterations in cell metabolism, impairment of migratory capacity, and sensitization of DBTRG cells to a chemotherapeutic drug, the dichloroacetate (DCA). Taken together, our findings provide evidence that the miR-144 plus DCA combined therapy holds promise to overcome GB-acquired chemoresistance, therefore deserving to be explored toward its potential application as a complementary therapeutic approach to the current treatment options for this type of brain tumor.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , MicroARNs/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Metabolismo Energético , Perfilación de la Expresión Génica , Glioblastoma/metabolismo , Humanos , ARN Mensajero/genéticaRESUMEN
PURPOSE: This study aimed to endow the cell-penetrating peptide (CPP) S413-PV with adequate features towards a safe and effective application in cancer gene therapy. METHODS: Peptide/siRNA complexes were prepared with two new derivatives of the CPP S413-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S413-PV peptide, being named C12-H5-S413-PV and H5-S413-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum. RESULTS: Peptide/siRNA complexes prepared with the C12-H5-S413-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H5-S413-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes. CONCLUSIONS: These encouraging results pave the way for a potential application of the C12-H5-S413-PV peptide as a promising tool in cancer gene therapy.
Asunto(s)
Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Silenciador del Gen , Terapia Genética/métodos , Histidina/química , Ácidos Láuricos/química , Neoplasias/genética , Neoplasias/terapia , Péptidos/química , Péptidos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Humanos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Estearoil-CoA Desaturasa/antagonistas & inhibidoresRESUMEN
Glioblastoma (GBM) is a deadly and therapy resistant malignant brain tumour, characterized by an aggressive and diffuse growth pattern, which prevents complete surgical resection. Despite advances in the identification of genomic and molecular alterations that fuel the tumour, average patient survival post-diagnosis remains very low (â¼14.6-months). In addition to being highly heterogeneous, GBM tumour cells exhibit high adaptive capacity to targeted molecular therapies owing to an established network of signalling cascades with functional redundancy, which provides them with robust compensatory survival mechanisms. Here, we investigated whether a multimodal strategy combining multitargeted tyrosine kinase inhibitors (MTKIs) and microRNA (miRNA) modulation could overcome the signalling pathway redundancy in GBM and, hence, promote tumour cell death. By performing a high-throughput screening, we identified a myriad of miRNAs, including those belonging to the miR-302-3p/372-3p/373-3p/520-3p family, which coordinately act with the MTKI sunitinib to decrease GBM cell viability. Two members of this family, hsa-miRNA-302a-3p and hsa-miRNA-520 b, were found to modulate the expression of receptor tyrosine kinase mediators (including AKT1, PIK3CA and SOS1) in U87 and DBTRG human GBM cells. Importantly, administration of mimics of these miRNAs with sunitinib or axitinib resulted in decreased tumour cell proliferation and enhanced cell death, whereas no significant effect was observed when coupling miRNA modulation with temozolomide, the first-line drug for GBM therapy. Overall, our results provide evidence that combining the 'horizontal' inhibition of signalling pathways promoted by MTKIs with the 'vertical' inhibition of the downstream signalling cascade promoted by hsa-miR-302a-3p and hsa-miR-520 b constitutes a promising approach towards GBM treatment.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , MicroARNs/genética , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Terapia Combinada , Predisposición Genética a la Enfermedad , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , MicroARNs/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , TransfecciónRESUMEN
Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.
Asunto(s)
Técnicas de Transferencia de Gen , Genes Mitocondriales , Compuestos de Amonio Cuaternario , Alquenos/química , Polarización de Fluorescencia , Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Lípidos de la Membrana/química , Plásmidos/administración & dosificación , Plásmidos/genética , Compuestos de Amonio Cuaternario/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Tensoactivos/químicaRESUMEN
Menadione (MEN), a polycyclic aromatic ketone, was shown to promote cell injury by imposing massive oxidative stress and has been proposed as a promising chemotherapeutic agent for the treatment of cancer diseases. The mechanisms underlying MEN-induced mitochondrial dysfunction and cell death are not yet fully understood. In this work, a systematic study was performed to unveil the effects of MEN on membrane lipid organization, using models mimicking mitochondrial membranes and native mitochondrial membranes. MEN was found to readily incorporate in membrane systems composed of a single phospholipid (phosphatidylcholine) or the lipids dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and tetraoleoylcardiolipin at 1:1:1 molar ratio, as well as in mitochondrial membranes. Increased permeability in both membrane models, monitored by calcein release, seemed to correlate with the extent of MEN incorporation into membranes. MEN perturbed the physical properties of vesicles composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine plus tetraoleoylcardiolipin (at 7:3 molar ratio), as reflected by the downshift of the lipid phase transition temperature and the emergence of a new transition peak in the mixed lipid system, detected by DSC. (31)P NMR studies revealed that MEN favored the formation of non-lamellar structures. Also, quenching studies with the fluorescent probes DPH and TMA-DPH showed that MEN distributed across the bilayer thickness in both model and native mitochondrial membranes. MEN's ability to promote alterations of membrane lipid organization was related with its reported mitochondrial toxicity and promotion of apoptosis, predictably involved in its anti-carcinogenic activity.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Membranas Artificiales , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Fosfatidiletanolaminas/metabolismo , Vitamina K 3/metabolismo , Biofisica , Rastreo Diferencial de Calorimetría , Permeabilidad de la Membrana Celular , Fluoresceínas/metabolismo , Fluorescencia , Humanos , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Mitocondrias/química , Membranas Mitocondriales/química , Fosfatidiletanolaminas/química , Espectrofotometría , Vitamina K 3/químicaRESUMEN
Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3-acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Carcinoma de Células Escamosas/radioterapia , Proliferación Celular/efectos de la radiación , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/radioterapia , Lutecio/uso terapéutico , Radioinmunoterapia , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Proliferación Celular/efectos de los fármacos , Cetuximab , Receptores ErbB/inmunología , Femenino , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Lutecio/farmacocinética , Ratones , Ratones Endogámicos BALB C , Panitumumab , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.
Asunto(s)
Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Membrana Dobles de Lípidos/química , Péptidos/química , Péptidos/farmacocinética , Membrana Celular/química , Membrana Celular/ultraestructura , Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos/métodos , Células HeLa , Humanos , Membrana Dobles de Lípidos/metabolismo , Péptidos/farmacología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismoRESUMEN
Triterpenoids have multiple biological properties, although little information is available regarding their toxicity. The present study evaluates the toxicity of two new synthetic lupane derivatives using distinct biological models including synthetic lipids membranes, isolated liver and heart mitochondria fractions, and cell lines in culture. The two novel triterpenoids caused perturbations in the organization of synthetic lipid bilayers, leading to changes in membrane fluidity. Inhibition of cell proliferation and mitochondrial and nuclear morphological alterations were also identified. Inhibition of mitochondrial oxygen consumption, transmembrane electric potential depolarization and induction of the mitochondrial permeability transition pore was observed, although effects on isolated mitochondrial fractions were tissue-dependent (e.g. liver vs. heart). The size and length of hydrocarbon chains in the two molecules appear to be determinant for the degree of interaction with mitochondria, especially in the whole cell environment, where more barriers for diffusion exist. The results suggest that the positively charged triterpenoids target mitochondria and disrupt bioenergetics.
Asunto(s)
Membrana Dobles de Lípidos/antagonistas & inhibidores , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Modelos Biológicos , Triterpenos/toxicidad , Animales , Aniones/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Membrana Dobles de Lípidos/metabolismo , Masculino , Mitocondrias Cardíacas/química , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Conformación Molecular , Ratas , Ratas Wistar , Triterpenos/químicaRESUMEN
BACKGROUND: Cell-penetrating peptides (CPPs) have been extensively exploited in gene therapy approaches as vectors for intracellular delivery of bioactive molecules. The ability of CPPs to be internalized into cells and their capacity to complex nucleic acids depend on their molecular structure, both primary and secondary, namely regarding hydrophobicity/hydrophilicity. CPP acylation has been used as a strategy to improve this structural feature. METHODS: Acyl groups (from 6 to 18 carbon atoms) were attached to the S413-PV peptide and their effects on the peptide competence to complex siRNAs and to mediate gene silencing in glioblastoma (GBM) cells were studied. A systematic characterization of membrane interactions with S413-PV acyl-derivatives was also conducted, using different biophysical techniques (surface pressure-area isotherms in Langmuir monolayers, DSC and 31P NMR) to unravel a relationship between CPP biological activity and CPP effects on membrane stability and lipid organization. RESULTS: A remarkable concordance was noticed between acylated-S413-PV peptide competence to promote gene silencing in GBM cells and disturbance induced in membrane models, the lauroyl- and myristoyl-S413-PV peptides being the most effective. A cut-off effect was described for the first time regarding the influence of acyl-chain length on CPP bioactivity. CONCLUSIONS: C12-S413-PV showed high capacity to destabilize lipid bilayers, to escape from lysosomal degradation and to mediate gene silencing without promoting cytotoxicity. GENERAL SIGNIFICANCE: Besides unraveling a new CPP with high potential to be employed as a gene delivery vector, this work emphasizes the benefit from allying biophysical and biological studies towards a proper CPP structural refinement for successful pre-clinical/clinical application.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Metabolismo de los Lípidos , Ácidos Nucleicos/administración & dosificación , Péptidos/metabolismo , Acilación , Línea Celular Tumoral , Humanos , Membrana Dobles de Lípidos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Ácidos Nucleicos/metabolismo , TransfecciónRESUMEN
The occurrence of protein synthesis errors (mistranslation) above the typical mean mistranslation level of 10-4 is mostly deleterious to yeast, zebrafish and mammal cells. Previous yeast studies have shown that mistranslation affects fitness and deregulates genes related to lipid metabolism, but there is no experimental proof that such errors alter yeast lipid profiles. We engineered yeast strains to misincorporate serine at alanine and glycine sites on a global scale and evaluated the putative effects on the lipidome. Lipids from whole cells were extracted and analysed by thin layer chromatography (TLC), liquid chromatography-mass spectrometry(LC-MS) and gas chromatography (GC). Oxidative damage, fatty acid desaturation and membrane fluidity changes were screened to identify putative alterations in lipid profiles in both logarithmic (fermentative) and post-diauxic shift (respiratory) phases. There were alterations in several lipid classes, namely lyso-phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and triglyceride, and in the fatty acid profiles, namely C16:1, C16:0, C18:1 and C18:0. Overall, the relative content of lipid species with saturated FA increased in detriment of those with unsaturated fatty acids. The expression of the OLE1 mRNA was deregulated, but phospholipid fluidity changes were not observed. These data expand current knowledge of mistranslation biology and highlight its putative roles in human diseases.
Asunto(s)
Ácidos Grasos/metabolismo , Biosíntesis de Proteínas , Saccharomyces cerevisiae/metabolismo , Estearoil-CoA Desaturasa/biosíntesis , Ácidos Grasos/genética , Saccharomyces cerevisiae/genética , Estearoil-CoA Desaturasa/genéticaRESUMEN
Glioblastoma (GBM), the highest grade astrocytoma, is one of the most aggressive and challenging cancers to treat. The standard treatment is usually limited due to the intrinsic resistance of GBM to chemotherapy and drug non-specific effects. Therefore, new therapeutic strategies need to be developed to target tumor cells, sparing healthy tissues. In this context, the inhibitor-of-apoptosis protein (IAP) survivin emerges as an ideal target for a gene silencing approach, since it is sharply differentially expressed in cancer tissues. In this work, two different families of cationic gemini surfactants (bis-quat conventional and serine-derived) were tested regarding their efficiency to deliver small interfering RNAs (siRNAs) in a human GBM cell line (U87), in order to select an effective siRNA anti-survivin carrier. Importantly, survivin downregulation combined with administration of the chemotherapeutic agents temozolomide or etoposide resulted in a synergistic cytotoxic effect, thus revealing to be a promising strategy to reduce the chemotherapeutic doses for GBM treatment.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Proteínas Inhibidoras de la Apoptosis/genética , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Regulación hacia Abajo , Silenciador del Gen , Glioblastoma/genética , Humanos , SurvivinRESUMEN
Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems.
Asunto(s)
ADN/química , ADN/genética , Compuestos de Amonio Cuaternario/química , Serina/química , Tensoactivos/química , Amidas/química , Aminas/química , Línea Celular Tumoral , Química Farmacéutica/métodos , Ésteres/química , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Células HeLa , Humanos , Lípidos/química , Transfección/métodosRESUMEN
The traditional view of mitochondria as cell powerhouses is a matter of common knowledge, but the overall view of these extraordinary organelles has been revolutionized in the last years. In fact, a large number of important and diverse processes take place at the mitochondrial level, which clearly surpass the energy production scope, intruding the critical fragile balance between cell life and death. The entangled biochemistry of mitochondrial membranes has been found to be dependent on specific lipid requirements, with cardiolipin holding a great part of the raised functional interest. Mitochondria contain a complex membrane system, based on a variety of lipids and exquisite asymmetries. Mitochondria lipid membrane composition depends on a tight interplay with the endoplasmic reticulum, from which some of the lipids present in the mitochondrial membranes have to be imported, at least in the form of precursors. Here, we review some external interventions resulting in alterations of mitochondrial lipid content, namely dietary interventions and genetic manipulation. Such manipulations of mitochondrial membrane lipid composition should result in physiological impact, given the importance of lipid-protein interactions within the mitochondrial membrane boundaries. We provide arguments for future experiments using the most modern chemical and biophysical approaches as well as computer simulation studies applied to appropriate biological membrane model systems, in order to identify the effects exerted by diet-induced lipid changes on membrane physical properties.
Asunto(s)
Lípidos de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Animales , Biología Computacional , Dieta , Humanos , Lípidos de la Membrana/genéticaRESUMEN
Gemini surfactants have been successfully used as components of gene delivery systems. In the present work, a family of gemini surfactants, represented by the general structure [CmH2m+1(CH3)2N(+)(CH2)sN(+)(CH3)2CmH2m+1]2Br(-), or simply m-s-m, was used to prepare cationic gene carriers, aiming at their application in transfection studies. An extensive characterization of the gemini surfactant-based complexes, produced with and without the helper lipids cholesterol and DOPE, was carried out in order to correlate their physico-chemical properties with transfection efficiency. The most efficient complexes were those containing helper lipids, which, combining amphiphiles with propensity to form structures with different intrinsic curvatures, displayed a morphologically labile architecture, putatively implicated in the efficient DNA release upon complex interaction with membranes. While complexes lacking helper lipids were translocated directly across the lipid bilayer, complexes containing helper lipids were taken up by cells also by macropinocytosis. This study contributes to shed light on the relationship between important physico-chemical properties of surfactant-based DNA vectors and their efficiency to promote gene transfer, which may represent a step forward to the rational design of gene delivery systems.
Asunto(s)
Membrana Celular/química , Técnicas de Transferencia de Gen/instrumentación , Tensoactivos/química , Supervivencia Celular , Química Física , ADN/química , Células HeLa , Humanos , Estructura Molecular , PlásmidosRESUMEN
The increasing knowledge on the genetic basis of disease has allowed the development of promising gene-targeted therapies that can be applied to numerous diseases. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or repair and regulation of specific genes. However, the clinical application of these large and charged molecules remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. Since the first observations, two decades ago, that some protein-derived domains can translocate across biological membranes, a wide group of peptides called cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. The mechanistic basis of CPP and CPP conjugate cellular uptake remains controversial. However, biophysical studies on the interactions of CPPs with membrane models have contributed to unravel the mechanisms underlying CPP membrane translocation as well as to propose relationships between those mechanisms and CPP efficiency in mediating cargo delivery. In this review, representative examples of CPPs were gathered from the most recent literature in order to emphasize the contributions of chemists, biophysicists and cell biologists towards the rational design of increasingly more efficient delivery systems. In this context, the present review aims at giving an overview of some of the most significant CPP families and their biological applications as nucleic acid delivery systems.