Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 181
Filtrar
1.
Sci Rep ; 10(1): 343, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31941923

RESUMEN

During intercellular communication, cells release extracellular vesicles such as exosomes, which contain proteins, ncRNAs and mRNAs that can influence proliferation and/or trigger apoptosis in recipient cells, and have been proposed to play an essential role in promoting invasion of tumor cells and in the preparation of metastatic niches. Our group proposed the antisense non-coding mitochondrial RNA (ASncmtRNA) as a new target for cancer therapy. ASncmtRNA knockdown using an antisense oligonucleotide (ASO-1537S) causes massive death of tumor cells but not normal cells and strongly reduces metastasis in mice. In this work, we report that exosomes derived from ASO-1537S-treated MDA-MB-231 breast cancer cells (Exo-1537S) inhibits tumorigenesis of recipient cells, in contrast to exosomes derived from control-ASO-treated cells (Exo-C) which, in contrast, enhance these properties. Furthermore, an in vivo murine peritoneal carcinomatosis model showed that Exo-1537S injection reduced tumorigenicity compared to controls. Proteomic analysis revealed the presence of Lactadherin and VE-Cadherin in exosomes derived from untreated cells (Exo-WT) and Exo-C but not in Exo-1537S, and the latter displayed enrichment of proteasomal subunits. These results suggest a role for these proteins in modulation of tumorigenic properties of exosome-recipient cells. Our results shed light on the mechanisms through which ASncmtRNA knockdown affects the preparation of breast cancer metastatic niches in a peritoneal carcinomatosis model.


Asunto(s)
Exosomas/metabolismo , Mitocondrias/genética , ARN no Traducido/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Superficie/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Proteínas de la Leche/metabolismo , Oligorribonucleótidos Antisentido/metabolismo , Oligorribonucleótidos Antisentido/farmacología , ARN no Traducido/antagonistas & inhibidores , ARN no Traducido/genética , Trasplante Heterólogo
2.
J Orthop Surg Res ; 14(1): 305, 2019 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-31492154

RESUMEN

BACKGROUND: Staphylococcus aureus (S. aureus) has the potential to opportunistically cause infectious diseases, including osteomyelitis, skin infections, pneumonia, and diarrhea. We previously reported that ASyycG RNA reduced the transcripts of virulent genes, and biofilm formation of S. aureus. Currently, graphene oxide (GO) nanosheets are used to efficiently deliver nucleic acids with favorable biocompatibility. METHODS: In the current study, a GO-based recombinant pDL278 ASyycG vector transformation strategy was developed. The particle size distributions and zeta-potential of the GO-PEI-based ASyycG were evaluated. The ASyycG plasmids were labeled with gene-encoding enhanced green fluorescent protein (ASyycG-eGFP). Quantitative real-time PCR assays were performed to investigate the expression of yycF/G/H and icaADB genes. Biofilm biomass and bacterial viability of S. aureus were evaluated by scanning electron microscopy and confocal laser scanning microscopy. We found that the expression of the yycG gene was inversely correlated with levels of the ASyycG transcripts and that the GO-PEI-ASyycG strain had the lowest expression of biofilm organization-associated genes. RESULTS: The results showed that the GO-based strategy significantly increased ASyycG transformation as a delivery system compared to the conventional competence-stimulating peptide strategy. Furthermore, GO-PEI-ASyycG suppressed bacterial biofilm aggregation and improved bactericidal effects on S. aureus after 24 h biofilm establishment. CONCLUSIONS: Our findings demonstrated that nano-GO with antisense yycG RNA is a more effective and relatively stable strategy for the management of S. aureus infections.


Asunto(s)
Antibacterianos/farmacología , Grafito/farmacología , ARN sin Sentido/farmacología , Staphylococcus aureus/efectos de los fármacos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Supervivencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Vectores Genéticos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Nanoestructuras , Oligorribonucleótidos Antisentido/farmacología , Tamaño de la Partícula , Staphylococcus aureus/genética , Staphylococcus aureus/fisiología
3.
Cell Metab ; 27(4): 714-739, 2018 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-29617640

RESUMEN

RNA-targeted therapies represent a platform for drug discovery involving chemically modified oligonucleotides, a wide range of cellular RNAs, and a novel target-binding motif, Watson-Crick base pairing. Numerous hurdles considered by many to be impassable have been overcome. Today, four RNA-targeted therapies are approved for commercial use for indications as diverse as Spinal Muscular Atrophy (SMA) and reduction of low-density lipoprotein cholesterol (LDL-C) and by routes of administration including subcutaneous, intravitreal, and intrathecal delivery. The technology is efficient and supports approaching "undruggable" targets. Three additional agents are progressing through registration, and more are in clinical development, representing several chemical and structural classes. Moreover, progress in understanding the molecular mechanisms by which these drugs work has led to steadily better clinical performance and a wide range of mechanisms that may be exploited for therapeutic purposes. Here we summarize the progress, future challenges, and opportunities for this drug discovery platform.


Asunto(s)
Terapia Molecular Dirigida , Atrofia Muscular Espinal/terapia , Oligorribonucleótidos Antisentido/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Animales , Descubrimiento de Drogas , Terapia Genética , Humanos , Oligorribonucleótidos Antisentido/química , Oligorribonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología
4.
Neuropharmacology ; 135: 11-21, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29510185

RESUMEN

It is well known that Wnt5a activation plays a pivotal role in brain injury and ß-arrestin2 induces c-Jun N-terminal kinase (JNK3) activation is involved in neuronal cell death. Nonetheless, the relationship between Wnt5a and JNK3 remains unexplored during cerebral ischemia/reperfusion (I/R). In the present study, we tested the hypothesis that Wnt5a-mediated JNK3 activation via the Wnt5a-Dvl-1-ß-arrestin2-JNK3 signaling pathway was correlated with I/R brain injury. We found that cerebral I/R could enhance the assembly of the Dvl-1-ß-arrestin2-JNK3 signaling module, Dvl-1 phosphorylation and JNK3 activation. Activated JNK3 could phosphorylate the transcription factor c-Jun, prompt caspase-3 activation and ultimately lead to neuronal cell death. To further explore specifically Wnt5a mediated JNK3 pathway activation in neuronal injury, we used Foxy-5 (a peptide that mimics the effects of Wnt5a) and Box5 (a Wnt5a antagonist) both in vitro and in vivo. AS-ß-arrestin2 (an antisense oligonucleotide against ß-arrestin2) and RRSLHL (a small peptide that competes with ß-arrestin2 for binding to JNK3) were applied to confirm the positive signal transduction effect of the Dvl-1-ß-arrestin2-JNK3 signaling module during cerebral I/R. Furthermore, Box5 and the RRSLHL peptide were found to play protective roles in neuronal death both in vivo global and focal cerebral I/R rat models and in vitro oxygen glucose deprivation (OGD) neural cells. In summary, our results indicate that Wnt5a-mediated JNK3 activation participates in I/R brain injury by targeting the Dvl-1-ß-arrestin2/JNK3 interaction. Our results also point to the possibility that disrupting Wnt5a-JNK3 signaling pathway may provide a new approach for stroke therapy.


Asunto(s)
Región CA1 Hipocampal/metabolismo , Proteínas Dishevelled/metabolismo , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Neuroprotección , Daño por Reperfusión/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Wnt-5a/metabolismo , Arrestina beta 2/metabolismo , Animales , Región CA1 Hipocampal/citología , Muerte Celular/efectos de los fármacos , Masculino , Neuroprotección/efectos de los fármacos , Oligopéptidos/farmacología , Oligorribonucleótidos Antisentido/farmacología , Péptidos/farmacología , Fosforilación , Ratas , Daño por Reperfusión/patología , Proteína Wnt-5a/agonistas , Arrestina beta 2/antagonistas & inhibidores
6.
J Crohns Colitis ; 11(2): 221-228, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27484097

RESUMEN

BACKGROUND AND AIMS: Carbohydrate sulphotransferase 15 [CHST15] is a specific enzyme biosynthesizing chondroitin sulphate E that binds various pathogenic mediators and is known to create local fibrotic lesions. We evaluated the safety of STNM01, a synthetic double-stranded RNA oligonucleotide directed against CHST15, in Crohn's disease [CD] patients whose mucosal lesions were refractory to conventional therapy. METHODS: This was a randomized, double-blind, placebo-controlled, concentration-escalation study of STNM01 by a single-dose endoscopic submucosal injection in 18 CD patients. Cohorts of increasing concentration of STNM01 were enrolled sequentially as 2.5nM [n = 3], 25nM [n = 3], and 250nM [n = 3] were applied. A cohort of placebo [n = 3] was included in each concentration. Safety was monitored for 30 days. Pharmacokinetics was monitored for 24h. The changes from baseline in the segmental Simple Endoscopic Score for CD [SES-CD] as well as the histological fibrosis score were evaluated. RESULTS: STNM01 was well tolerated and showed no drug-related adverse effects in any cohort of treated patients. There were no detectable plasma concentrations of STNM01 at all measured time points in all treatment groups. Seven of nine subjects who received STNM01 showed reduction in segmental SES-CD at Day 30, when compared with those who received placebo. Histological analyses of biopsy specimens revealed that STNM01 reduced the extent of fibrosis. CONCLUSION: Local application of STNM01 is safe and well tolerated in CD patients with active mucosal lesions.


Asunto(s)
Sulfatos de Condroitina , Enfermedad de Crohn , Mucosa Intestinal , Glicoproteínas de Membrana , ARN Interferente Pequeño/farmacología , Sulfotransferasas , Biopsia/métodos , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/metabolismo , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/patología , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas/métodos , Resección Endoscópica de la Mucosa/métodos , Femenino , Fibrosis , Fármacos Gastrointestinales/farmacología , Humanos , Inyecciones Intralesiones , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Gravedad del Paciente , Sulfotransferasas/antagonistas & inhibidores , Sulfotransferasas/metabolismo , Resultado del Tratamiento
7.
Proc Natl Acad Sci U S A ; 113(43): 11998-12005, 2016 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-27790981

RESUMEN

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.


Asunto(s)
Antiprotozoarios/farmacología , Leishmaniasis Mucocutánea/tratamiento farmacológico , Leishmaniavirus/efectos de los fármacos , Oligorribonucleótidos Antisentido/farmacología , ARN Bicatenario/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , Animales , Antiprotozoarios/química , Antiprotozoarios/metabolismo , Expresión Génica , Secuencias Invertidas Repetidas , Leishmania braziliensis/patogenicidad , Leishmania braziliensis/virología , Leishmania guyanensis/patogenicidad , Leishmania guyanensis/virología , Leishmaniasis Mucocutánea/parasitología , Leishmaniasis Mucocutánea/virología , Leishmaniavirus/genética , Leishmaniavirus/metabolismo , Macrófagos/parasitología , Macrófagos/virología , Ratones , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/metabolismo , Interferencia de ARN/efectos de los fármacos , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Simbiosis/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Replicación Viral/efectos de los fármacos
8.
J Neurosci ; 36(28): 7415-27, 2016 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-27413152

RESUMEN

UNLABELLED: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. SIGNIFICANCE STATEMENT: α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.


Asunto(s)
Cuerpos de Inclusión/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación/genética , Neuronas/metabolismo , Transcitosis/fisiología , alfa-Sinucleína/metabolismo , Animales , Regulación de la Expresión Génica/genética , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligorribonucleótidos Antisentido/farmacología , Fotoblanqueo , Ratas , Sinucleínas/metabolismo , Transcitosis/genética , Tubulina (Proteína)/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismo
9.
Curr Opin Microbiol ; 33: 47-55, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27375107

RESUMEN

Antisense antimicrobial therapeutics are synthetic oligomers that silence expression of specific genes. This specificity confers an advantage over broad-spectrum antibiotics by avoiding unintended effects on commensal bacteria. The sequence-specificity and short length of antisense antimicrobials also pose little risk to human gene expression. Because antisense antimicrobials are a platform technology, they can be rapidly designed and synthesized to target almost any microbe. This reduces drug discovery time, and provides flexibility and a rational approach to drug development. Recent work has shown that antisense technology has the potential to address the antibiotic-resistance crisis, since resistance mechanisms for standard antibiotics apparently have no effect on antisense antimicrobials. Here, we describe current reports of antisense antimicrobials targeted against viruses, parasites, and bacteria.


Asunto(s)
Infecciones Bacterianas/tratamiento farmacológico , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligorribonucleótidos Antisentido/farmacología , Enfermedades Parasitarias/tratamiento farmacológico , Oligonucleótidos Fosforotioatos/farmacología , Virosis/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Antivirales/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Descubrimiento de Drogas/métodos , Parásitos/efectos de los fármacos , Parásitos/genética , Virus/efectos de los fármacos , Virus/genética
10.
Differentiation ; 90(4-5): 91-100, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26677981

RESUMEN

MicroRNAs (miRNAs) are critical in the maintenance, differentiation, and lineage commitment of stem cells. Stem cells have the unique property to differentiate into tissue-specific cell types (lineage commitment) during cell division (self-renewal). In this study, we investigated whether miR-34a, a cell cycle-regulating microRNA, could control the stem cell properties of adipose tissue-derived stem cells (ADSCs). First, we found that the expression level of miR-34a was increased as the cell passage number was increased. This finding, however, was inversely correlated with our finding that the overexpression of miR-34a induced the decrease of cell proliferation. In addition, miR-34a overexpression decreased the expression of various cell cycle regulators such as CDKs (-2, -4, -6) and cyclins (-E, -D), but not p21 and p53. The cell cycle analysis showed accumulation of dividing cells at S phase by miR-34a, which was reversible by co-treatment with anti-miR-34a. The potential of adipogenesis and osteogenesis of ADSCs was also decreased by miR-34a overexpression, which was recovered by co-treatment with anti-miR-34a. The surface expression of stem cell markers including CD44 was also down-regulated by miR-34a overexpression as similar to that elicited by cell cycle inhibitors. miR-34a also caused a significant decrease in mRNA expression of stem cell transcription factors as well as STAT-3 expression and phosphorylation. Cytokine profiling revealed that miR-34a significantly modulated IL-6 and -8 production, which was strongly related to cellular senescence. These data suggest the importance of miR-34a for the fate of ADSCs toward senescence rather than differentiation.


Asunto(s)
Adipogénesis/genética , Ciclo Celular/genética , Senescencia Celular/genética , MicroARNs/genética , Factor de Transcripción STAT3/metabolismo , Células Madre/citología , Tejido Adiposo/citología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/genética , Humanos , Receptores de Hialuranos/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , MicroARNs/antagonistas & inhibidores , Oligorribonucleótidos Antisentido/farmacología , Osteogénesis/genética , Células Madre/fisiología
11.
Am J Hum Genet ; 97(4): 555-66, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26411495

RESUMEN

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a "podocyte-injury model" as the pathomechanism for SRNS due to biallelic NUP107 mutations.


Asunto(s)
Edad de Inicio , Mutación/genética , Síndrome Nefrótico/congénito , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Animales , Células Cultivadas , Niño , Preescolar , Citoplasma/metabolismo , Femenino , Haplotipos , Humanos , Immunoblotting , Inmunoprecipitación , Lactante , Riñón/metabolismo , Riñón/patología , Masculino , Microscopía Fluorescente , Síndrome Nefrótico/etiología , Síndrome Nefrótico/patología , Poro Nuclear , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Oligorribonucleótidos Antisentido/farmacología , Linaje , Podocitos/metabolismo , Podocitos/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/antagonistas & inhibidores
12.
Blood ; 126(15): 1844-55, 2015 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-26286849

RESUMEN

Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide "gapmer" to Berkeley SCD mice to selectively reduce circulating FII levels to ∼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FII(lox/-) mice (constitutively carrying ∼10% normal FII) and FII(WT) mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies.


Asunto(s)
Anemia de Células Falciformes/complicaciones , Terapia Genética , Hipertensión Pulmonar/prevención & control , Inflamación/prevención & control , Protrombina/fisiología , Enfermedades Vasculares/prevención & control , Anemia de Células Falciformes/mortalidad , Anemia de Células Falciformes/fisiopatología , Animales , Coagulación Sanguínea , Células Cultivadas , Hipertensión Pulmonar/etiología , Técnicas para Inmunoenzimas , Inflamación/etiología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Oligorribonucleótidos Antisentido/farmacología , Protrombina/antagonistas & inhibidores , Tasa de Supervivencia , Trombina/metabolismo , Enfermedades Vasculares/etiología
13.
Mol Cancer Res ; 13(6): 1009-21, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25758165

RESUMEN

UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Xenoinjertos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/química , Invasividad Neoplásica , Oligorribonucleótidos Antisentido/uso terapéutico
14.
Int J Oncol ; 46(3): 1169-80, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25585941

RESUMEN

Immune evasion of cancer cells is mainly due to the impaired transduction of apoptotic signals from immune cells to cancer cells, as well as inhibition of subsequent apoptosis signal cascades within the cancer cells. Over the past few decades, the research has focused more on the impaired transduction of the apoptotic signal from immune cells to cancer cells, rather than inhibition of the intracellular signaling pathways. In this study, miR­221 inhibitor was transfected into bladder cancer cell lines 5637, J82 and T24 to repress the expression of miR­221. As a result, the repression of miR­221 on p53 upregulated modulator of apoptosis (PUMA) was abolished, resulting in increased expression of the pro-apoptotic Bax and reduced expression of the anti-apoptotic Bcl-2, which promotes apoptosis of bladder cancer cells. The expression of MMP-2, MMP-9 and VEGF-C were reduced, resulting in reduced invasiveness and infiltration capability of bladder cancer cells, thereby inhibiting the immune evasion of bladder cancer cells.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , MicroARNs/fisiología , Proteínas Proto-Oncogénicas/genética , Escape del Tumor/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/inmunología , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , MicroARNs/antagonistas & inhibidores , Invasividad Neoplásica , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN/efectos de los fármacos , Interferencia de ARN/fisiología , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/patología , Proteína X Asociada a bcl-2/genética
15.
Mol Oncol ; 8(8): 1429-40, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24974076

RESUMEN

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent.


Asunto(s)
Proteína BRCA2/metabolismo , Cisplatino/farmacología , Animales , Proteína BRCA2/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Embrión de Pollo , Humanos , Metástasis de la Neoplasia/genética , Oligorribonucleótidos Antisentido/farmacología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo
16.
J Endocrinol ; 217(2): 131-40, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23349329

RESUMEN

The 72 kDa inositol polyphosphate 5-phosphatase E (72k-5ptase) controls signal transduction through the catalytic dephosphorylation of the 5-position of membrane-bound phosphoinositides. The reduction of 72k-5ptase expression in the hypothalamus results in improved hypothalamic insulin signal transduction and reduction of food intake and body mass. Here, we evaluated the tissue distribution and the impact of obesity on the expression of 72k-5ptase in peripheral tissues of experimental animals. In addition, insulin signal transduction and action were determined in an animal model of obesity and insulin resistance treated with an antisense (AS) oligonucleotide that reduces 72k-5ptase expression. In lean Wistar rats, 72k-5ptase mRNA and protein are found in highest levels in heart, skeletal muscle, and white adipose tissue. In three distinct models of obesity, Wistar rats, Swiss mice fed on high-fat diet, and leptin-deficient ob/ob mice, the expression of 72k-5ptase is increased in skeletal muscle and adipose tissue. The treatment of obese Wistar rats with an anti-72k-5ptase AS oligonucleotide results in significant reduction of 72k-5ptase catalytic activity, which is accompanied by reduced food intake and body mass and improved insulin signal transduction and action as determined by immunoblotting and clamp studies respectively. 72k-5ptase expression is increased in obesity and its AS inhibition resulted in a significant improvement in insulin signal transduction and restoration of glucose homeostasis.


Asunto(s)
Dieta Alta en Grasa/efectos adversos , Insulina/fisiología , Obesidad/etiología , Obesidad/fisiopatología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Transducción de Señal/fisiología , Tejido Adiposo Blanco/enzimología , Animales , Modelos Animales de Enfermedad , Inositol Polifosfato 5-Fosfatasas , Resistencia a la Insulina/fisiología , Leptina/deficiencia , Masculino , Ratones , Ratones Obesos , Músculo Esquelético/enzimología , Miocardio/enzimología , Obesidad/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Monoéster Fosfórico Hidrolasas/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Ratas Wistar
17.
Recent Pat DNA Gene Seq ; 7(1): 74-81, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23061527

RESUMEN

Protein kinase C (PKC) comprises a family of 10 serine/threonine kinases divided into 3 subfamilies: classical, novel and atypical. These isoenzymes represent one of the major mediators of signal transduction, and most may be associated with several pathogenic processes including malignant transformation or cancer and metastasis. Moreover, some activated isoenzymes are also involved in other diseases such as infarct, rejection due to incomplete histocompatibility in organ transplantation, pain, diabetic macular edema, etc. Here, we review several patents related to inhibitors of PKC that represent a new and promising strategy for the prevention and treatment of these illnesses. Among these inhibitors, we included antisense oligonucleotides as another useful strategy to treat infectious and autoimmune diseases associated with misregulated expression of PKC and tumour necrosis factor alpha (TNF-α). On the other hand, two different activators of PKC and their applications related to neurodegenerative diseases have also been reviewed in this work.


Asunto(s)
Activadores de Enzimas/farmacología , Patentes como Asunto , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Axones/efectos de los fármacos , Biomarcadores de Tumor , Neoplasias Encefálicas/enzimología , Activadores de Enzimas/uso terapéutico , Humanos , Infarto del Miocardio/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Oligorribonucleótidos Antisentido/farmacología , Dolor/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Trasplantes
18.
PLoS One ; 7(10): e47690, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23110089

RESUMEN

EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , División Celular/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Diseño de Fármacos , Oligorribonucleótidos Antisentido/farmacología , División del ARN/efectos de los fármacos , Ribonucleasa P/metabolismo , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Microscopía Confocal , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Regiones Terminadoras Genéticas/genética
19.
Hum Gene Ther ; 23(12): 1313-8, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22992134

RESUMEN

Collagen VI gene mutations cause Ullrich and Bethlem muscular dystrophies. Pathogenic mutations frequently have a dominant negative effect, with defects in collagen VI chain secretion and assembly. It is agreed that, conversely, collagen VI haploinsufficiency has no pathological consequences. Thus, RNA-targeting approaches aimed at preferentially inactivating the mutated COL6 messenger may represent a promising therapeutic strategy. By in vitro studies we obtained the preferential depletion of the mutated COL6A2 messenger, by targeting a common single-nucleotide polymorphism (SNP), cistronic with a dominant COL6A2 mutation. We used a 2'-O-methyl phosphorothioate (2'OMePS) antisense oligonucleotide covering the SNP within exon 3, which is out of frame. Exon 3 skipping has the effect of depleting the mutated transcript via RNA nonsense-mediated decay, recovering the correct collagen VI secretion and restoring the ability to form an interconnected microfilament network into the extracellular matrix. This novel RNA modulation approach to correcting dominant mutations may represent a therapeutic strategy potentially applicable to a great variety of mutations and diseases.


Asunto(s)
Colágeno Tipo VI/genética , Distrofias Musculares/genética , Mutación , Oligorribonucleótidos Antisentido/farmacología , Células Cultivadas , Colágeno Tipo VI/metabolismo , Exones , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/patología , Genes Dominantes , Humanos , Degradación de ARNm Mediada por Codón sin Sentido , Polimorfismo de Nucleótido Simple
20.
J Leukoc Biol ; 92(4): 859-67, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22730547

RESUMEN

Patients with 10-30 days postburn injury are greatly susceptible to infections. M1M (IL-10(-)IL-12(+) M) are essential cells in host antibacterial innate immunity against MRSA infections. However, these effector cells are not easily generated in hosts who are carriers of M2bM (IL-12(-)IL-10(+)CCL1(+)LIGHT(+) M). M2bM are inhibitory on M1M generation. In this study, the antibacterial resistance of mice, 10-30 days postburn injury against MRSA infection, was improved by the modulation of M2bM activities. Unburned mice inoculated with M preparations from mice, 10-30 days after burn injury, were susceptible to MRSA infection, whereas unburned mice, inoculated with M preparations from the same mice that were previously treated with CCL1 antisense ODN, were resistant to the infection. M2bM, isolated from Day 15 burn mice, lost their M2bM properties 3 days after cultivation under frequent medium changes, whereas their M2bM properties remained in the same cultures supplemented with rCCL1. In cultures, M preparations from Day 15 burn mice treated with CCL1 antisense ODN did not produce CCL1 and did convert to M1M after heat-killed MRSA stimulation. Also, Day 15 burn mice treated with the ODN became resistant against MRSA infection. These results indicate that CCL1 released from M2bM is essentially required for the maintenance of their properties. The increased susceptibility of mice, 10-30 days after burn injury to MRSA infection, may be controlled through the intervention of CCL1 production by M2bM appearing in association with severe burn injuries.


Asunto(s)
Quimiocina CCL1/fisiología , Macrófagos/inmunología , Animales , Quemaduras/microbiología , Interleucina-10/biosíntesis , Masculino , Staphylococcus aureus Resistente a Meticilina , Ratones , Ratones Endogámicos BALB C , Oligorribonucleótidos Antisentido/farmacología , Infecciones Estafilocócicas/inmunología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...