RESUMEN
Studying metabolism may assist in understanding the relationship between normal and dysfunctional mitochondrial activity and various diseases, such as neurodegenerative, cardiovascular, autoimmune, psychiatric, and cancer. Nuclear magnetic resonance-based metabolomics represents a powerful method to characterize the chemical content of complex samples and has been successfully applied to studying a range of conditions. However, an optimized methodology is lacking for analyzing isolated organelles, such as mitochondria. In this study, we report the development of a protocol to metabolically profile mitochondria from healthy, tumoral, and metastatic tissues. Encouragingly, this approach provided quantitative information about up to 45 metabolites in one comprehensive and robust analysis. Our results revealed significant differences between whole-cell and mitochondrial metabolites, which supports a more refined approach to metabolic analysis. We applied our optimized methodology to investigate aggressive and metastatic breast cancer in mouse tissues, discovering that lung mitochondria exhibit an altered metabolic fingerprint. Specific amino acids, organic acids, and lipids showed significant increases in levels when compared with mitochondria from healthy tissues. Our optimized methodology could promote a better understanding of the molecular mechanisms underlying breast cancer aggressiveness and mitochondrial-related diseases and support the optimization of new advanced therapies.
Asunto(s)
Mitocondrias , Neoplasias , Ratones , Animales , Mitocondrias/metabolismo , Metabolómica/métodos , Neoplasias/metabolismo , Espectroscopía de Resonancia Magnética , Aminoácidos/metabolismoRESUMEN
Alcohol abuse induces the expression of inflammatory mediators by activating the immune receptors to trigger neuroinflammation and brain damage; however, therapies that reduce neuroimmune system activation may protect against alcohol's damaging effects. Curcuminoids possess anti-inflammatory properties but suffer from low bioavailability; therefore, we designed a new receptor-targeted biodegradable star-shaped crosslinked polypeptide polymer that bears propargylamine moieties and bisdemethoxycurcumin (StClPr-BDMC-ANG) as an enhanced anti-inflammatory therapeutic that penetrates the blood-brain-barrier and ameliorates alcohol-induced neuroinflammation. StClPr-BDMC-ANG administration maintains the viability of primary glia and inhibits the ethanol-induced upregulation of crucial inflammatory mediators in the prefrontal and medial cortex in a mouse model of chronic ethanol consumption. StClPr-BDMC-ANG treatment also suppresses the ethanol-mediated downregulation of microRNAs known to negatively modulate neuroinflammation in the brain cortex (miRs 146a-5p and let-7b-5p). In summary, our results demonstrate the attenuation of alcohol-induced neuroinflammation by an optimized and targeted polypeptide-based nanoconjugate of a curcuminoid.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Curcumina/análogos & derivados , Nanoconjugados/administración & dosificación , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Péptidos/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Curcumina/administración & dosificación , Curcumina/química , Ratones , Nanoconjugados/química , Enfermedades Neuroinflamatorias/inducido químicamente , Péptidos/químicaRESUMEN
Triple-negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA-MB-231-Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer-associated adipocyte infiltration in the MDA-MB-231-Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA-MB-231-Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA-MB-231-Luc model. Additionally, we discovered ß-immunoglobulinemia and increased basal levels of G-CSF correlating with a metastatic switch, with G-CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread.
Asunto(s)
Modelos Animales de Enfermedad , Neoplasias Mamarias Experimentales/terapia , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Femenino , Humanos , Metástasis Linfática , Neoplasias Mamarias Experimentales/diagnóstico , Neoplasias Mamarias Experimentales/metabolismo , Metabolómica/métodos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Reproducibilidad de los Resultados , Neoplasias de la Mama Triple Negativas/diagnóstico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The high incidence of prostate carcinogenesis has prompted the search for novel effective treatment approaches. We have employed curcumin (Curc) and diethylstilbestrol (DES) to synthesize a series of polyacetal (PA)-based combination conjugates for prostate cancer (PCa) treatment. Given their bihydroxyl functionalities, Curc and DES molecules were incorporated into a PA mainchain using a one-pot reaction between diols and divinyl ethers. The PA-conjugates released both drugs under acidic conditions, such as those found in the tumor microenvironment, endosomes, or lysosomes, while remaining stable at neutral pH 7.4. The drug ratio was optimized to achieve anticancer drug synergism with elevated cytotoxicity against LNCaP-hormone-dependent human PCa cells conferred via the induction of S phase cell cycle arrest by the upregulation of p53 and CDK inhibitors p21Waf/CIP1 and downregulation of cyclin D1. The application of rationally designed PA-Curc-DES combination conjugates represents a potentially exciting new treatment for prostate cancer.
Asunto(s)
Acetales/química , Antineoplásicos , Curcumina/química , Dietilestilbestrol/química , Polímeros/química , Neoplasias de la Próstata/tratamiento farmacológico , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Preparaciones de Acción Retardada/síntesis química , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
PURPOSE: miRNAs are noncoding RNAs that negatively regulate target mRNA gene expression. Aberrant miRNA expression is associated with prostate cancer pathogenesis. We identified miRNAs as potential biomarkers for prostate cancer diagnosis and prognosis. MATERIALS AND METHODS: Total RNA was obtained from 10 normal prostate and 50 prostate cancer samples, and analyzed using the GeneChip® miRNA 2.0 Array. At a median followup of 92 months (range 2 to 189) an independent cohort of 273 paraffin embedded prostate cancer samples was used for validation by quantitative reverse transcriptase-polymerase chain reaction. Another 92 urine samples from patients undergoing prostate biopsy were evaluated for these miRNAs. RESULTS: miR-182 and 187, the miRNAs most differentially expressed between normal and tumor tissue, were selected for further validation. miR-187 inversely correlated with cT (p = 0.125) and pT (p = 0.0002) stages, Gleason score (p = 0.003) and TMPRSS2-ERG status (p = 0.003). The log rank test showed associations of miR-182 with biochemical (p = 0.026) and clinical (p = 0.043) progression-free survival, as also noted on multivariate analysis. A significant independent improvement in the definition of risk of progression was achieved by combining miR-182 expression with Gleason score (p <0.0001). miR-187 detection in urine provided an independent predictive value for positive biopsy. A prediction model including serum prostate specific antigen, urine PCA3 and miR-187 provided 88.6% sensitivity and 50% specificity (AUC 0.711, p = 0.001). CONCLUSIONS: Results show that miR-182 and 187 are promising biomarkers for prostate cancer prognosis to identify patients at risk for progression and for diagnosis to improve the predictive capability of existing biomarkers.
Asunto(s)
Biomarcadores de Tumor , MicroARNs , Prostatectomía , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/cirugía , Anciano , Biomarcadores de Tumor/orina , Diagnóstico Precoz , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , MicroARNs/orina , Pronóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/orina , Estudios RetrospectivosRESUMEN
Modifying biological agents with polymers such as polyethylene glycol (PEG) has demonstrated clinical benefits; however, post-market surveillance of PEGylated derivatives has revealed PEG-associated toxicity issues, prompting the search for alternatives. We explore how conjugating a poly-l-glutamic acid (PGA) to an anti-insulin growth factor 1 receptor antibody (AVE1642) modulates the bio-nano interface and anti-tumor activity in preclinical prostate cancer models. Native and PGA-modified AVE1642 display similar anti-tumor activity in vitro; however, AVE1642 prompts IGF-1R internalization while PGA conjugation prompts higher affinity IGF-1R binding, thereby inhibiting IGF-1R internalization and altering cell trafficking. AVE1642 attenuates phosphoinositide 3-kinase signaling, while PGA-AVE1642 inhibits phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. PGA conjugation also enhances AVE1642's anti-tumor activity in an orthotopic prostate cancer mouse model, while PGA-AVE1642 induces more significant suppression of cancer cell proliferation/angiogenesis than AVE1642. These findings demonstrate that PGA conjugation modulates an antibody's bio-nano interface, mechanism of action, and therapeutic activity.
Asunto(s)
Ácido Glutámico , Neoplasias de la Próstata , Animales , Ratones , Masculino , Humanos , Fosfatidilinositol 3-Quinasas , Neoplasias de la Próstata/tratamiento farmacológico , Proliferación Celular , Fosfatidilinositol 3-Quinasa , PolietilenglicolesRESUMEN
Prostate cancer (PCa), one of the leading causes of cancer-related deaths, currently lacks effective treatment for advanced-stage disease. Paclitaxel (PTX) is a highly active chemotherapeutic drug and the first-line treatment for PCa; however, conventional PTX formulation causes severe hypersensitivity reactions and limits PTX use at high concentrations. In the pursuit of high molecular weight, biodegradable, and pH-responsive polymeric carriers, one conjugates PTX to a polyacetal-based nanocarrier to yield a tert-Ser-PTX polyacetal conjugate. tert-Ser-PTX conjugate provides sustained release of PTX over 2 weeks in a pH-responsive manner while also obtaining a degree of epimerization of PTX to 7-epi-PTX. Serum proteins stabilize tert-Ser-PTX, with enhanced stability in human serum versus PBS (pH 7.4). In vitro efficacy assessments in PCa cells demonstrate IC50 values above those for the free form of PTX due to the differential cell trafficking modes; however, in vivo tolerability assays demonstrate that tert-Ser-PTX significantly reduces the systemic toxicities associated with free PTX treatment. tert-Ser-PTX also effectively inhibits primary tumor growth and hematologic, lymphatic, and coelomic dissemination, as confirmed by in vivo and ex vivo bioluminescence imaging and histopathological evaluations in mice carrying orthotopic LNCaP tumors. Overall, the results suggest the application of tert-Ser-PTX as a robust antitumor/antimetastatic treatment for PCa.
Asunto(s)
Antineoplásicos Fitogénicos , Neoplasias de la Próstata , Acetales , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Portadores de Fármacos/química , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Paclitaxel/química , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Polímeros/química , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O(2)) as somatic cells. We hypothesized that O(2) levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O(2)) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O(2) levels; after 24 hours at 5% O(2), more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Oxígeno/metabolismo , Angiopoyetina 1/genética , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Hipoxia de la Célula/genética , Trasplante de Células/métodos , Células Cultivadas , Regulación hacia Abajo/genética , Células Madre Embrionarias/citología , Células Endoteliales/citología , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Infarto del Miocardio/cirugía , Neovascularización Fisiológica/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Ratas , Ratas Desnudas , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Even given recent advances in nanomedicine development of breast cancer treatment in recent years and promising results in pre-clinical models, cancer nanomedicines often fail at the clinical trial stage. Limitations of conventional in vitro models include the lack of representation of the stromal population, the absence of a three-dimensional (3D) structure, and a poor representation of inter-tumor and intra-tumor heterogeneity. Herein, we review those cell culture strategies that aim to overcome these limitations, including cell co-cultures, advanced 3D cell cultures, patient-derived cells, bioprinting, and microfluidics systems. The in vivo evaluation of nanomedicines must consider critical parameters that include the enhanced permeability and retention effect, the host's immune status, and the site of tumor implantation. Here, we critically discuss the advantages and limitations of current in vivo models and report how the improved selection and application of breast cancer models can improve the clinical translation of nanomedicines.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Desarrollo de Medicamentos , Nanomedicina , Nanopartículas/química , Antineoplásicos/química , Neoplasias de la Mama/patología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Femenino , HumanosRESUMEN
Treatment of triple negative breast cancer (TNBC)-associated metastasis represents an unmet clinical need, and we lack effective therapeutics for a disease that exhibits high relapse rates and associates with poor patient outcomes. Advanced nanosized drug delivery systems may enhance the efficacy of first-line chemotherapeutics by altering drug pharmacokinetics and enhancing tumor/metastasis targeting to significantly improve efficacy and safety. Herein, we propose the application of injectable poly-amino acid-based nanogels (NGs) as a versatile hydrophilic drug delivery platform for the treatment of TNBC lung metastasis. We prepared biocompatible and biodegradable cross-linked NGs from polyglutamic acid (PGA) loaded with the chemotherapeutic agent doxorubicin (DOX). Our optimized synthetic procedures generated NGs of ~100 nm in size and 25 wt% drug loading content that became rapidly internalized in TNBC cell lines and displayed IC50 values comparable to the free form of DOX. Importantly, PGA-DOX NGs significantly inhibited lung metastases and almost completely suppressed lymph node metastases in a spontaneously metastatic orthotopic mouse TNBC model. Overall, our newly developed PGA-DOX NGs represent a potentially effective therapeutic strategy for the treatment of TNBC metastases.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Animales , Línea Celular Tumoral , Doxorrubicina/uso terapéutico , Portadores de Fármacos/uso terapéutico , Humanos , Ratones , Nanogeles , Ácido Poliglutámico/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Human dental pulp contains precursor cells termed dental pulp stem cells (DPSC) that show self-renewal and multilineage differentiation and also secrete multiple proangiogenic and antiapoptotic factors. To examine whether these cells could have therapeutic potential in the repair of myocardial infarction (MI), DPSC were infected with a retrovirus encoding the green fluorescent protein (GFP) and expanded ex vivo. Seven days after induction of myocardial infarction by coronary artery ligation, 1.5 x 10(6) GFP-DPSC were injected intramyocardially in nude rats. At 4 weeks, cell-treated animals showed an improvement in cardiac function, observed by percentage changes in anterior wall thickening left ventricular fractional area change, in parallel with a reduction in infarct size. No histologic evidence was seen of GFP+ endothelial cells, smooth muscle cells, or cardiac muscle cells within the infarct. However, angiogenesis was increased relative to control-treated animals. Taken together, these data suggest that DPSC could provide a novel alternative cell population for cardiac repair, at least in the setting of acute MI.
Asunto(s)
Pulpa Dental/citología , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Neovascularización Fisiológica , Trasplante de Células Madre , Células Madre/citología , Función Ventricular Izquierda/fisiología , Adolescente , Adulto , Animales , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Pulpa Dental/trasplante , Pulpa Dental/ultraestructura , Pulpa Dental/virología , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/citología , Miocitos del Músculo Liso/citología , Ratas , Ratas Desnudas , Retroviridae , Infecciones por Retroviridae , Células Madre/ultraestructura , Células Madre/virología , UltrasonografíaRESUMEN
The intrinsic characteristics of the tumor microenvironment (TME), including acidic pH and overexpression of hydrolytic enzymes, offer an exciting opportunity for the rational design of TME-drug delivery systems (DDS). We developed and characterized a pH-responsive biodegradable poly-L-glutamic acid (PGA)-based combination conjugate family with the aim of optimizing anticancer effects. We obtained combination conjugates bearing Doxorubicin (Dox) and aminoglutethimide (AGM) with two Dox loadings and two different hydrazone pH-sensitive linkers that promote the specific release of Dox from the polymeric backbone within the TME. Low Dox loading coupled with a short hydrazone linker yielded optimal effects on primary tumor growth, lung metastasis (â¼90% reduction), and toxicological profile in a preclinical metastatic triple-negative breast cancer (TNBC) murine model. The use of transcriptomic analysis helped us to identify the molecular mechanisms responsible for such results including a differential immunomodulation and cell death pathways among the conjugates. This data highlights the advantages of targeting the TME, the therapeutic value of polymer-based combination approaches, and the utility of -omics-based analysis to accelerate anticancer DDS.
Asunto(s)
Antineoplásicos/administración & dosificación , Portadores de Fármacos/química , Ácido Poliglutámico/química , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Microambiente Tumoral , Aminoglutetimida/administración & dosificación , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Liberación de Fármacos , Femenino , Xenoinjertos , Humanos , Concentración de Iones de Hidrógeno , Ratones Endogámicos BALB C , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Metabolomics is becoming a relevant tool for understanding the molecular mechanisms involved in the response to new drug delivery systems. The applicability of this experimental approach to cell cultures and animal models makes metabolomics a useful tool for establishing direct connections between in vitro and in vivo data, thus providing a reliable platform for the characterization of chemotherapeutic agents. Herein, we used metabolomic profiles based on nuclear magnetic resonance (NMR) spectroscopy to evaluate the biochemical pathways involved in the response to a chemotherapeutic anthracycline drug (Doxorubicin, Dox) and an N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-conjugated form (HPMA-Dox) in an in vitro cell culture model and an in vivo orthotopic breast cancer model. We also used protein expression and flow cytometry studies to obtain a better coverage of the biochemical alterations associated with the administration of these compounds. The overall analysis revealed that polymer conjugation leads to increased apoptosis, reduced glycolysis, and reduced levels of phospholipids when compared to the free chemotherapeutic drug. Our results represent a first step in the application of integrated in vitro and in vivo metabolomic studies to the evaluation of drug delivery systems.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Metabolómica/métodos , Polímeros/química , Animales , Western Blotting , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Células MCF-7 , Espectroscopía de Resonancia Magnética , Ratones , Nanomedicina , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In this study, we have employed 1H NMR metabolomics to assess the metabolic responses of PC3 prostate tumour cells to hypoxia and to pharmacological HIF-1α inhibition by DES or its polyacetal conjugate tert-DES. Oxygen deprivation prompted a number of changes in intracellular composition and metabolic activity, mainly reflecting upregulated glycolysis, amino acid catabolism and other compensatory mechanisms used by hypoxic cells to deal with oxidative imbalance and energy deficit. Cell treatment with a non-cytotoxic concentration of DES, under hypoxia, triggered significant changes in 17 metabolites. Among these, lactate, phosphocreatine and reduced glutathione, whose levels showed opposite variations in hypoxic and drug-treated cells, emerged as possible markers of DES-induced HIF-1α inhibition. Furthermore, the free drug had a much higher impact on the cellular metabolome than tert-DES, particularly concerning polyamine and pyrimidine biosynthetic pathways, known to be tightly involved in cell proliferation and growth. This is likely due to the different cell pharmacokinetics observed between free and conjugated DES. Overall, this study has revealed a number of unanticipated metabolic changes that inform on DES and tert-DES direct cellular effects, providing further insight into their mode of action at the biochemical level.
Asunto(s)
Acetales/farmacología , Dietilestilbestrol/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Metabolómica/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Polímeros/farmacología , Neoplasias de la Próstata , Acetales/química , Acetales/uso terapéutico , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Dietilestilbestrol/química , Dietilestilbestrol/uso terapéutico , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Polímeros/química , Polímeros/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Due to the polyanionic nature of DNA, typically cationic or neutral delivery vehicles have been used for gene delivery. As a new approach, this study focuses on the design, development, and validation of nonviral polypeptide-based carriers for oligonucleotide delivery based on a negatively charged poly-l-glutamic acid (PGA) backbone partly derivatized with oligoaminoamide residues. To this end, PGA-derivatives modified with different pentameric succinyl tetraethylene pentamines (Stp5 ) are designed. Optionally, histidines for modulation of endosomal buffer capacity and cysteines for pDNA complex stabilization are included, followed by characterization of biophysical properties and gene transfer efficiency in N2a neuroblastoma or 4T1 breast cancer cells.
Asunto(s)
Amidas/química , ADN/genética , Etilenodiaminas/química , Técnicas de Transferencia de Gen , Ácido Poliglutámico/química , Línea Celular Tumoral , Cisteína/química , ADN/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histidina/química , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Neuronas/metabolismo , Neuronas/patología , Plásmidos/química , Plásmidos/metabolismo , Ácido Poliglutámico/metabolismo , Succinimidas/químicaRESUMEN
The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous "ordinary-extraordinary" phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery.
Asunto(s)
Péptidos/química , Sistemas de Liberación de Medicamentos , Nanomedicina , Nanoestructuras , Ácido PoliglutámicoRESUMEN
miRNAs are predicted to control the activity of approximately 60% of all protein-coding genes participating in the regulation of several cellular processes and diseases, including cancer. Recently, we have demonstrated that miR-187 is significantly downregulated in prostate cancer (PCa) and here we propose a proteomic approach to identify its potential targets. For this purpose, PC-3 cells were transiently transfected with miR-187 precursor and miRNA mimic negative control. Proteins were analyzed by a two-dimensional difference gel electrophoresis (2D-DIGE) and defined as differentially regulated if the observed fold change was ±1.06. Then, MALDI-TOF MS analysis was performed after protein digestion and low abundance proteins were identified by LC-MS/MS. Peptides were identified by searching against the Expasy SWISS PROT database, and target validation was performed both in vitro by western blot and qRT-PCR and in clinical samples by qRT-PCR, immunohistochemistry and ELISA. DIGE analysis showed 9 differentially expressed spots (p<0.05) and 7 showed a down-regulated expression upon miR-187 re-introduction. Among these targets we identified aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3 expression was significantly downregulated in PC3, LNCaP and DU-145 cells after miR-187 re-introduction. Supporting these data, the expression of ALDH1A3 was found significantly (p<0.0001) up-regulated in PCa samples and inversely correlated (p<0.0001) with miR-187 expression, its expression being directly associated with Gleason score (p = 0.05). The expression of ALDH1A3 was measured in urine samples to evaluate the predictive capability of this biomarker for the presence of PCa and, at a signification level of 10%, PSA and also ALDH1A3 were significantly associated with a positive biopsy of PCa. In conclusion, our data illustrate for the first time the role of ALDH1A3 as a miR-187 target in PCa and provide insights in the utility of using this protein as a new biomarker for PCa.
Asunto(s)
Aldehído Oxidorreductasas/genética , MicroARNs/fisiología , Neoplasias de la Próstata/genética , Aldehído Oxidorreductasas/metabolismo , Biomarcadores de Tumor/orina , Línea Celular Tumoral , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/enzimología , Interferencia de ARNRESUMEN
The design of improved polymeric carriers to be used in the next generation of polymer therapeutics is an ongoing challenge. Biodegradable systems present potential advantages regarding safety benefit apart from the possibility to use higher molecular weight (Mw) carriers allowing PK optimization, by exploiting the enhanced permeability and retention (EPR)-mediated tumor targeting. Within this context, we previously designed pH-responsive polyacetalic systems, tert-polymers, where a drug with the adequate diol-functionality was incorporated within the polymer mainchain. The synthetic, non-steroidal estrogen, diethylstilboestrol (DES) clinically used for the treatment of advanced prostate cancer was chosen as drug. In order to improve the properties of this tert-polymer, novel polyacetalic systems as block-co-polymers, with more defined structure have been obtained. This second generation polyacetals allowed higher drug capacity than the tert-polymer, a biphasic DES release profile at acidic pH and due to its controlled amphiphilic character readily formed micelle-like structures in solution. These features result in an enhancement of conjugate therapeutic value in selected prostate cancer cell models. Exhaustive physico-chemical characterization focusing on nanoconjugate solution behavior and using advanced techniques, such as, pulsed-gradient spin-echo NMR (PGSE-NMR) and small-angle neutron scattering (SANS), has been carried out in order to demonstrate this hypothesis. Clear evidence of significantly different conformation in solution has been obtained for both polyacetals. These results demonstrate that an adequate control on molecular or supramolecular conformation in solution with polymer therapeutics is crucial in order to achieve the desired therapeutic output.
Asunto(s)
Acetales/síntesis química , Antineoplásicos/síntesis química , Dietilestilbestrol/síntesis química , Portadores de Fármacos/síntesis química , Diseño de Fármacos , Polímeros/síntesis química , Neoplasias de la Próstata/tratamiento farmacológico , Acetales/química , Acetales/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dietilestilbestrol/administración & dosificación , Dietilestilbestrol/química , Dietilestilbestrol/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Estabilidad de Medicamentos , Endocitosis , Humanos , Concentración de Iones de Hidrógeno , Masculino , Microscopía Confocal , Conformación Molecular , Peso Molecular , Polímeros/química , Polímeros/farmacología , Neoplasias de la Próstata/patología , Solubilidad , Soluciones , EstereoisomerismoRESUMEN
In order to explore the influence of polymer microstructure and stereochemistry in biological settings, the synthesis, micellization, cellular fate and the use in paclitaxel formulations of poly(N-(2-hydroxypropyl)-methacrylamide)-block-poly(L-lactide) (P(HPMA)-block-P(LLA)) and poly(N-(2-hydroxypropyl)-methacrylamide)-block-poly(DL-lactide) block copolymers (P(HPMA)-block-P(DLLA)) were studied. To this end, P(HPMA)-block-P(lactide) block copolymers and their fluorescently labeled analogues were synthesized. The polymers exhibited molecular weights M(n) around 20,000 g/mol with dispersities (D=M(w)/M(n)) below 1.3. In addition, the solution conformation of this new type of partially degradable amphiphilic block copolymers was studied with and without paclitaxel loading in PBS buffer (pH 7.2), employing fluorescence correlation spectroscopy (FCS). We observed polymeric micelles with a hydrodynamic diameter of 17.0 nm for a fluorescently labeled P(HPMA)-block-P(LLA) block copolymer (P2*) and 20.4 nm for a P(HPMA)-block-P(DLLA) block copolymer (P3*). For the corresponding loaded block copolymers aggregates with a diameter of 40.0 nm (P2*) and 41.4 nm (P3*) in formulations containing 17 wt.% paclitaxel were observed, respectively. While the block copolymer itself showed non-toxic behavior up to a concentration of 3 mg/mL in HeLa (human cervix adenocarcinoma) cells, the paclitaxel containing formulations showed IC 50 values in the range of 10-100 nM. The P(HPMA)-block-P(DLLA) polymer (P3*) enters the cells more efficiently than stereo regular polymer (P2*) via an energy-dependent uptake mechanism. Thus, differences in the IC(50) value are--most likely--attributed to significant changes in cellular uptake. Polymer tacticity and stereoregularity appear to represent a key feature determining cellular uptake and efficiency for the PLA block copolymer drug formulations. This work demonstrates the importance of the microstructure of polymers used in drug delivery systems (DDS).