RESUMEN

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Colagenasas/farmacología , Nanopartículas/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colágeno/química , Colágeno/genética , Colagenasas/química , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/genética , Fibrosis/tratamiento farmacológico , Fibrosis/patología , Fibrosis/prevención & control , Humanos , Liposomas/química , Liposomas/farmacología , Ratones , Nanopartículas/uso terapéutico , Paclitaxel/química , Paclitaxel/farmacología , Páncreas/efectos de los fármacos , Páncreas/patología , Microambiente Tumoral/efectos de los fármacos

RESUMEN

Novel bioconjugates (Agm6-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped "head" (Agm6-M) to a polymeric "tail" (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm6-M-PEG-FA and Agm6-M-PEG-OCH3, respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm6-M-PEG-OCH3:Agm6-M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size <80 nm with narrow polydispersity and rod-shaped morphology. The polyplexes were stable for several hours in plasma while ON was released in the presence of heparin concentration 16-times higher than the physiological one. The polyplexes displayed negligible cytotoxicity, hemolysis and low pro-inflammatory TNF-α release. Studies performed with FR-overexpressing HeLa and MDA-MB-231 cells using siRac1 revealed that the folated polyplexes caused significantly higher gene silencing (86.1 ±â€¯9.6%) and inhibition of cell migration (40%) than the non-folated polyplexes obtained with Agm6-M-PEG-OCH3 only. Although cytofluorimetric analyses showed similar cell uptake for both folated and non-folated polyplexes, confocal, TEM and competition studies showed that the folated polyplexes were taken-up by lysosome escaping caveolin-mediated pathway with final polyplex localization within cytosol, while non-folated polyplexes were preferentially taken-up via clathrin-mediated pathway to localize in the lysosomes. Finally, preliminary in vivo studies carried out in mice revealed that the folated polyplexes dispose in the tumor mass.

Asunto(s)

Transportadores de Ácido Fólico/metabolismo , Técnicas de Transferencia de Gen , Nanoconjugados/química , Oligonucleótidos/administración & dosificación , Animales , Unión Competitiva , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Transportadores de Ácido Fólico/genética , Silenciador del Gen , Células HeLa , Humanos , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Oligonucleótidos/genética , Tamaño de la Partícula , Unión Proteica , Propiedades de Superficie , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.

Asunto(s)

Neoplasias de la Mama/cirugía , Melanoma/cirugía , Nanopartículas/administración & dosificación , Imagen Óptica/métodos , Coloración y Etiquetado/métodos , Cirugía Asistida por Computador/métodos , Animales , Catepsinas/metabolismo , Modelos Animales de Enfermedad , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/metabolismo , Humanos , Ratones , Nanopartículas/metabolismo , Resultado del Tratamiento

RESUMEN

The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment might rely on simultaneous targeting of multiple genes, which can be achieved by RNA interference-based therapeutic strategies. Here we show a potent combination of microRNA and siRNA delivered by an efficient nanocarrier to PDAC tumors. Using proteomic-microRNA profiles and survival data of PDAC patients from TCGA, we found a novel signature for prolonged survival. Accordingly, we used a microRNA-mimic to increase miR-34a together with siRNA to silence PLK1 oncogene. For in vivo dual-targeting of this combination, we developed a biodegradable amphiphilic polyglutamate amine polymeric nanocarrier (APA). APA-miRNA-siRNA polyplexes systemically administered to orthotopically inoculated PDAC-bearing mice showed no toxicity and accumulated at the tumor, resulting in an enhanced antitumor effect due to inhibition of MYC oncogene, a common target of both miR-34a and PLK1. Taken together, our findings warrant this unique combined polyplex's potential as a novel nanotherapeutic for PDAC.

Asunto(s)

Carcinoma Ductal Pancreático/genética , Proteínas de Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Adulto , Anciano , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/terapia , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Portadores de Fármacos/química , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estimación de Kaplan-Meier , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Persona de Mediana Edad , Nanoestructuras/química , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/química , Tratamiento con ARN de Interferencia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Quinasa Tipo Polo 1

RESUMEN

RNA interference (RNAi) can contribute immensely to the area of personalized medicine by its ability to target any gene of interest. Nevertheless, its clinical use is limited by lack of efficient delivery systems. Polymer therapeutics can address many of the challenges encountered by the systemic delivery of RNAi, but suffer from inherent drawbacks such as polydispersity and batch to batch heterogeneity. These characteristics may have far-reaching consequences when dealing with therapeutic applications, as both the activity and the toxicity may be dependent on the length of the polymer chain. To investigate the consequences of polymers' heterogeneity, we have synthesized two batches of aminated poly(α)glutamate polymers (PGAamine), differing in their degree of polymerization, but not in the monomer units or their conjugation. Isothermal titration calorimetry study was conducted to define the binding affinity of these polymers with siRNA. Molecular dynamics simulation revealed that Short PGAamine:siRNA polyplexes exposed a higher amount of amine moieties to the surroundings compared to Long PGAamine. This resulted in a higher zeta potential, leading to faster degradation and diminished gene silencing. Altogether, our study highlights the importance of an adequate physico-chemical characterization to elucidate the structure⁻function-activity relationship, for further development of tailor-designed RNAi delivery vehicles.

RESUMEN

The expression of the CO2 -fixation enzyme ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which is affected by light, involves the cysteine-rich protein bundle-sheath defective-2 (BSD2) that was originally identified in maize bundle-sheath cells. We identified the BSD2 ortholog in Chlamydomonas reinhardtii as a small protein (17 kDa) localized to the chloroplast. The algal BSD2-ortholog contains four CXXCXGXG DnaJ-like elements, but lacks the other conserved domains of DnaJ. BSD2 co-migrated with the rbcL transcript on heavy polysomes, and both BSD2 and rbcL mRNA shifted to the lighter fractions under oxidizing conditions that repress the translation of the Rubisco large subunit (RbcL). This profile of co-migration supports the possibility that BSD2 is required for the de novo synthesis of RbcL. Furthermore, BSD2 co-migrated with the rbcL transcript in a C. reinhardtii premature-termination mutant that encodes the first 60 amino acids of RbcL. In both strains, BSD2 shared its migration profile with the rbcL transcript but not with psbA mRNA. The chaperone activity of BSD2 was exemplified by its ability to prevent the aggregation of both citrate synthase (CS) and RbcL in vitro following their chemical denaturation. This activity did not depend on the presence of the thiol groups on BSD2. In contrast, the activity of BSD2 in preventing the precipitation of reduced ß-chains in vitro in the insulin turbidity assay was thiol-dependent. We conclude that BSD2 combines a chaperone 'holdase' function with the ability to interact with free thiols, with both activities being required to protect newly synthesized RbcL chains.

Asunto(s)

Chlamydomonas reinhardtii/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/genética , Ribulosa-Bifosfato Carboxilasa/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estrés Oxidativo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Homología de Secuencia de Aminoácido , Sacarosa

RESUMEN

MicroRNAs (miRNAs) are small noncoding RNAs found to govern nearly every biological process. They frequently acquire a gain or a loss of function in cancer, hence playing a causative role in the development and progression of cancer. There are major obstacles on the way for the successful delivery of miRNA, which include low cellular uptake of the RNA and endosomal escape, immunogenicity, degradation in the bloodstream, and rapid renal clearance. The delivered miRNA needs to be successfully routed to the target organ, enter the cell and reach its intracellular target in an active form. Consequently, in order to exploit the promise of RNA interference, there is an urgent need for efficient methods to deliver miRNAs. These can be divided into three main categories: complexation, encapsulation, and conjugation. In this review, we will discuss the special considerations for miRNA delivery for cancer therapy, focusing on nonviral delivery systems: lipid, polymeric, and inorganic nanocarriers.

RESUMEN

For patients with primary lung cancer, accurate determination of the tumor type significantly influences treatment decisions. However, techniques and methods for lung cancer typing lack standardization. In particular, owing to limited tumor sample amounts and the poor quality of some samples, the classification of primary lung cancers using small preoperative biopsy specimens presents a diagnostic challenge using current tools. We previously described a microRNA-based assay (miRview squamous; Rosetta Genomics Ltd., Rehovot, Israel) that accurately differentiates between squamous and nonsquamous non-small cell lung cancer. Herein, we describe the development and validation of an assay that differentiates between the four main types of lung cancer: squamous cell carcinoma, nonsquamous non-small cell lung cancer, carcinoid, and small cell carcinoma. The assay, miRview lung (Rosetta Genomics Ltd.), is based on the expression levels of eight microRNAs, measured using a sensitive quantitative RT-PCR platform. It was validated on an independent set of 451 samples, more than half of which were preoperative cytologic samples (fine-needle aspiration and bronchial brushing and washing). The assay returned a result for more than 90% of the samples with overall accuracy of 94% (95% CI, 91% to 96%), with similar performance observed in pathologic and cytologic samples. Thus, miRview lung is a simple and reliable diagnostic assay that offers an accurate and standardized classification tool for primary lung cancer using pathologic and cytologic samples.

Asunto(s)

Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/diagnóstico , MicroARNs/genética , Técnicas de Diagnóstico Molecular/métodos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

There is emerging evidence for the prognostic role of various microRNA (miRNA) molecules in colon cancer. The aim of this study was therefore to compare the miRNA profiles in the primary tumor of patients with recurrent and non-recurrent colon cancer. The study population included 110 patients, 51 (46%) with stage I and 59 (54%) with stage II disease, who underwent curative colectomies between 1995 and 2005 without adjuvant therapy and for whom reliable miRNA expression data were available. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor samples. Initial profiling, using microarrays, was done in order to identify potential biomarkers of recurrence. The miRNA expression was later verified by quantitative real-time polymerase chain reaction (qRT-PCR). Findings were compared between patients who had a recurrence within 36 months of surgery (bad prognosis group, n=23, 21%) and those who did not (good prognosis group, n=87, 79%) in the entire group and within each stage. The results showed that in stage I, none of the 903 miRNAs tested showed differential expression between patients with good prognosis compared with those with poor prognosis. In contrast, in stage II, one miRNA, miR-29a, showed a clear differential expression between the groups (p=0.028). High expression of miR-29a was associated with a longer disease-free survival (DFS), on both univariate and multivariate analyses. Using miR-29a, the positive predictive value for non-recurrence was 94% (2 recurrences among 31 patients). The differential expression of miR-29a was verified by qRT-PCR, showing a similar impact of this miR on DFS. In conclusion, this study demonstrated a significant impact of miR-29a on the risk of recurrence in patients with stage II but not in patients with stage I colon cancer. Based on these results, a validation study is planned.

Asunto(s)

Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Expresión Génica , MicroARNs/genética , Recurrencia Local de Neoplasia/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Anciano , Anciano de 80 o más Años , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Sensibilidad y Especificidad

RESUMEN

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Asunto(s)

Biomarcadores de Tumor/genética , Mesotelioma , MicroARNs/genética , Análisis por Micromatrices/métodos , Neoplasias Pleurales , Regulación Neoplásica de la Expresión Génica , Humanos , Mesotelioma/diagnóstico , Mesotelioma/genética , Mesotelioma/patología , MicroARNs/metabolismo , Análisis por Micromatrices/normas , Neoplasias Pleurales/diagnóstico , Neoplasias Pleurales/genética , Neoplasias Pleurales/patología , Sensibilidad y Especificidad

RESUMEN

A recurring challenge for brain pathologists is to diagnose whether a brain malignancy is a primary tumor or a metastasis from some other tissue. The accurate diagnosis of brain malignancies is essential for selection of proper treatment. MicroRNAs are a class of small non-coding RNA species that regulate gene expression; many exhibit tissue-specific expression and are misregulated in cancer. Using microRNA expression profiling, we found that hsa-miR-92b and hsa-miR-9/hsa-miR-9* are over-expressed, specifically in brain primary tumors, as compared to primary tumors from other tissues and their metastases to the brain. By considering the expression of only these two microRNAs, it is possible to distinguish between primary and metastatic brain tumors with very high accuracy. These microRNAs thus represent excellent biomarkers for brain primary tumors. Previous reports have found that hsa-miR-92b and hsa-miR-9/hsa-miR-9* are expressed more strongly in developing neurons and brain than in adult brain. Thus, their specific over-expression in brain primary tumors supports a functional role for these microRNAs or a link between neuronal stem cells and brain tumorigenesis.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , MicroARNs/genética , Metástasis de la Neoplasia/diagnóstico , Adulto , Área Bajo la Curva , Diagnóstico Diferencial , Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Curva ROC , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad