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BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.
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Epithelial ovarian cancers (EOCs) are a heterogeneous group of tumors with different molecular and clinical features. In past decades, few improvements have been achieved in terms of EOC management and treatment efficacy, such that the 5-year survival rate of patients remained almost unchanged. A better characterization of EOCs' heterogeneity is needed to identify cancer vulnerabilities, stratify patients and adopt proper therapies. The mechanical features of malignant cells are emerging as new biomarkers of cancer invasiveness and drug resistance that can further improve our knowledge of EOC biology and allow the identification of new molecular targets. In this study, we determined the inter and intra-mechanical heterogeneity of eight ovarian cancer cell lines and their association with tumor invasiveness and resistance to an anti-tumoral drug with cytoskeleton depolymerization activity (2c).
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Antineoplásicos , Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Humanos , Femenino , Línea Celular Tumoral , Neoplasias Ováricas/metabolismo , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Biomarcadores de Tumor/metabolismoRESUMEN
BACKGROUND: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. METHODS: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. RESULTS: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. CONCLUSION: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.
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The treatment of urological cancers has been significantly improved in recent years. However, for the advanced stages of these cancers and/or for those developing resistance, novel therapeutic options need to be developed. Among the innovative strategies, the use of small interfering RNA (siRNA) seems to be of great therapeutic interest. siRNAs are double-stranded RNA molecules which can specifically target virtually any mRNA of pathological genes. For this reason, siRNAs have a great therapeutic potential for human diseases including urological cancers. However, the fragile nature of siRNAs in the biological environment imposes the development of appropriate delivery systems to protect them. Thus, ensuring siRNA reaches its deep tissue target while maintaining structural and functional integrity represents one of the major challenges. To reach this goal, siRNA-based therapies require the development of fine, tailor-made delivery systems. Polymeric nanoparticles, lipid nanoparticles, nanobubbles and magnetic nanoparticles are among nano-delivery systems studied recently to meet this demand. In this review, after an introduction about the main features of urological tumors, we describe siRNA characteristics together with representative delivery systems developed for urology applications; the examples reported are subdivided on the basis of the different delivery materials and on the different urological cancers.
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Cystic fibrosis (CF) is a disease characterized by the production of viscous mucoid secretions in multiple organs, particularly the airways. The pathological increase of proteins, mucin and biological polymers determines their arrangement into a three-dimensional polymeric network, affecting the whole mucus and impairing the muco-ciliary clearance which promotes inflammation and bacterial infection. Thus, to improve the efficacy of the drugs usually applied in CF therapy (e.g., mucolytics, anti-inflammatory and antibiotics), an in-depth understanding of the mucus nanostructure is of utmost importance. Drug diffusivity inside a gel-like system depends on the ratio between the diffusing drug molecule radius and the mesh size of the network. Based on our previous findings, we propose the combined use of rheology and low field NMR to study the mesh size distribution of the sputum from CF patients. Specifically, we herein explore the effects of chest physiotherapy on CF sputum characteristic as evaluated by rheology, low field NMR and the drug penetration through the mucus via mathematical simulation. These data show that chest physiotherapy has beneficial effects on patients, as it favourably modifies sputum and enhances drug penetration through the respiratory mucus.
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Fibrosis Quística , Nanoestructuras , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Humanos , Moco/metabolismo , Modalidades de Fisioterapia , Esputo/química , Esputo/metabolismoRESUMEN
BACKGROUND: As most cystic fibrosis (CF) patients progress to respiratory failure, lung functionality assessment is pivotal. We previously developed a test that indirectly monitors airways (inflammation/functional test) by measuring the spin-spin relaxation time (T2m) of the water hydrogens present in CF sputum. Here the T2m significance in the monitoring of CF lung disease was further investigated by studying the correlation of T2m with: 1) sputum viscoelasticity, 2) mucociliary clearability index (MCI)/cough clearability index (CCI) and 3) sputum average mesh-size. METHODS: Sputum samples from 25 consenting CF subjects were analyzed by rheology tests (elastic modulus G and zero shear viscosity η0) and Low Field Nuclear Magnetic (LF-NMR) resonance (T2m). MCI/CCI were calculated from the rheological parameters. The average mesh-size (ξ) of the sputum structure was then evaluated by rheology/LF-NMR, together with FEV1 for each patient. RESULTS: There was an inverse correlation between G and η0 versus T2m, indicating that a worsening of the lung condition (T2m-FEV1 drop) is paralleled by an increase in sputum viscoelasticity (G and η0) favoring mucus stasis/inflammation. A direct correlation was also observed between T2m and MCI/CCI, showing that T2m provides information as to airway mucus clearing. Moreover, there was a direct correlation between T2m and the average sputum mesh size (ξ). CONCLUSIONS: We demonstrated a correlation between T2m (measured in CF patient's sputum) and the sputum viscoelasticity/average mesh-size and with MCI/CCI, parameters related to airway mucus clearing. Thus, the present data strengthen the potential of our test to provide indirect monitoring of airway disease course in CF patients as T2m depends on mucus solid concentration and nanostructure.
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Fibrosis Quística/fisiopatología , Espectroscopía de Resonancia Magnética , Reología , Adulto , Anciano , Anciano de 80 o más Años , Fibrosis Quística/tratamiento farmacológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Depuración Mucociliar , Pruebas de Función Respiratoria , Esputo/química , ViscosidadRESUMEN
Hepatocellular carcinoma (HCC) is the sixth most common type of tumor and the second leading cause of tumor-related death worldwide. Liver cirrhosis is the most important predisposing factor for HCC. Available therapeutic approaches are not very effective, especially for advanced HCC, which is the most common form of the disease at diagnosis. New therapeutic strategies are therefore urgently needed. The use of animal models represents a relevant tool for preclinical screening of new molecules/strategies against HCC. However, several issues, including animal husbandry, limit the use of current models (rodent/pig). One animal model that has attracted the attention of the scientific community in the last 15 years is the zebrafish. This freshwater fish has several attractive features, such as short reproductive time, limited space and cost requirements for husbandry, body transparency and the fact that embryos do not show immune response to transplanted cells. To date, two different types of zebrafish models for HCC have been developed: the transgenic zebrafish and the zebrafish xenograft models. Since transgenic zebrafish models for HCC have been described elsewhere, in this review, we focus on the description of zebrafish xenograft models that have been used in the last five years to test new molecules/strategies against HCC.
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Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,ß-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes. Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay. Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects. In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Galactosa , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Ratones , Polímeros , ARN Interferente PequeñoRESUMEN
BACKGROUND: Ovary Carcinoma (OC) is the most lethal gynecological neoplasm due to the late diagnoses and to the common development of resistance to platinum-based chemotherapy. Thus, novel therapeutic approaches are urgently required. In this regard, the strategy of drug repurposing is becoming attractive. By this approach, the effectiveness of a drug originally developed for another indication is tested in a different pathology. The advantage is that data about pharmacokinetic properties and toxicity are already available. Thus, in principle, it is possible to reduce research costs and to speed up drug usage/marketing. RESULTS: Here, some noticeable examples of repurposed drugs for OC, such as amiodarone, ruxolitinib, statins, disulfiram, ormeloxifenem, and Quinacrine, are reported. Amiodarone, an antiarrhythmic agent, has shown promising anti-OC activity, although the systemic toxicity should not be neglected. The JAK inhibitor, Ruxolitinib, may be employed particularly in coadministration with standard OC therapy as it synergistically interacts with platinum-based drugs. Particularly interesting is the use of statin which represent one of the most commonly administered drugs in aged population to treat hypercholesterolemia. Disulfiram, employed in the treatment of chronic alcoholism, has shown anti-OC properties. Ormeloxifene, commonly used for contraception, seems to be promising, especially due to the negligible side effects. Finally, Quinacrine used as an antimicrobial and anti-inflammatory drug, is able to downregulate OC cell growth and promote cell death. CONCLUSION: Whereas further testing in patients are necessary to better clarify the therapeutic potential of repurposed drugs for OC, it is believed that their use, better if combined with OC targeted delivery systems, can significantly contribute to the development of novel and effective anti-OC treatments.
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Reposicionamiento de Medicamentos , Neoplasias Ováricas , Antineoplásicos/uso terapéutico , Disulfiram , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Preparaciones FarmacéuticasRESUMEN
PURPOSE: To develop a novel approach to monitor lung ventilation/inflammation in cystic fibrosis (CF) patients. Lung assessment in CF patients is relevant given that most patients succumb to respiratory failure. Respiratory functional tests (forced expiratory volume in the first second; FEV1 ) and inflammatory markers are used to test pulmonary ventilation/inflammation, respectively. However, FEV1 is effort dependent and might be uncomfortable for CF patients. Furthermore, inflammatory marker detection is costly and not rapid. To overcome these limitations, we propose the measurement, by means of low field nuclear magnetic resonance, of the spin-spin relaxation time (T2m ) of water hydrogens present in CF patient sputum. In CF sputum, different biological components are pathologically increased and inversely related to lung functionality. Moreover, we showed that these components alter in a dose-dependent manner the T2m in synthetic CF sputum. METHODS: Sputum samples were obtained from 42 CF subjects by voluntary expectoration; FEV1 , C-reactive protein (CRP), blood neutrophil counts together with cytokine (tumor necrosis factor alpha [TNFα], interleukin [IL]-1ß, IL-4, and vascular endothelial growth factor) quantifications were then evaluated. RESULTS: In sputum samples, we observe that T2m directly correlates (rFEV1 = 0.44; P < 10-4 ; 169 samples) with FEV1 . Moreover, T2m inversely correlates with the circulating inflammation markers CRP/neutrophil number (rCRP = -0.44, P < 10-4 ; rNC = -0.37, P < 2 * 10-4 ; 103 and 86 samples, respectively) and with the sputum inflammatory cytokines TNFα/IL-ß1 (rTNFα = -0.72, P < 10-4 ; rIL-1ß = -0.685, P < 10-4 ; 27 samples). T2m variations also correspond to FEV1 values over time in defined patients. CONCLUSION: These findings, together with the fast, reliable, and simple determination of T2m , make our approach a novel tool potentially usable in the real world of CF patients.
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Fibrosis Quística , Neumonía , Biomarcadores , Proteína C-Reactiva , Fibrosis Quística/diagnóstico por imagen , Citocinas , Humanos , Inflamación , Espectroscopía de Resonancia Magnética , Esputo , Factor A de Crecimiento Endotelial VascularRESUMEN
The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigation. Among the different novel drugs, small interfering RNAs (siRNAs) are particularly attractive because of their specificity of action and efficacy, as documented in many experimental setups. However, the fragility of these molecules in the biological environment necessitates the use of delivery materials able to protect them and possibly target them to the cancer cells. Among the different delivery materials, those based on polymers and lipids are considered very interesting because of their biocompatibility and ability to carry/deliver siRNAs. Despite these features, polymers and lipids need to be engineered to optimize their delivery properties for OC. In this review, we concentrated on the description of the therapeutic potential of siRNAs and polymer-/lipid-based delivery systems for OC. After a brief description of OC and siRNA features, we summarized the strategies employed to minimize siRNA delivery problems, the targeting strategies to OC, and the preclinical models available. Finally, we discussed the most interesting works published in the last three years about polymer-/lipid-based materials for siRNA delivery.
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Despite the advances in anticancer therapies, their effectiveness for many human tumors is still far from being optimal. Significant improvements in treatment efficacy can come from the enhancement of drug specificity. This goal may be achieved by combining the use of therapeutic molecules with tumor specific effects and delivery carriers with tumor targeting ability. In this regard, nucleic acid-based drug (NABD) and particularly small interfering RNAs (siRNAs), are attractive molecules due to the possibility to be engineered to target specific tumor genes. On the other hand, polymeric-based delivery systems are emerging as versatile carriers to generate tumor-targeted delivery systems. Here we will focus on the most recent findings in the selection of siRNA/polymeric targeted delivery systems for hepatocellular carcinoma (HCC), a human tumor for which currently available therapeutic approaches are poorly effective. In addition, we will discuss the most attracting and, in our opinion, promising siRNA-polymer combinations for HCC in relation to the biological features of HCC tissue. Attention will be also put on the mathematical description of the mechanisms ruling siRNA-carrier delivery, this being an important aspect to improve effectiveness reducing the experimental work.
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Carcinoma Hepatocelular , Sistemas de Liberación de Medicamentos/métodos , Neoplasias Hepáticas , Modelos Biológicos , Polímeros , ARN Interferente Pequeño , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Modelos Químicos , Polímeros/química , Polímeros/uso terapéutico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Because of its high biocompatibility, bio-degradability, low-cost and easy availability, cellulose finds application in disparate areas of research. Here we focus our attention on the most recent and attractive potential applications of cellulose in the biomedical field. We first describe the chemical/structural composition of cellulose fibers, the cellulose sources/features and cellulose chemical modifications employed to improve its properties. We then move to the description of cellulose potential applications in biomedicine. In this field, cellulose is most considered in recent research in the form of nano-sized particle, i.e., nanofiber cellulose (NFC) or cellulose nanocrystal (CNC). NFC is obtained from cellulose via chemical and mechanical methods. CNC can be obtained from macroscopic or microscopic forms of cellulose following strong acid hydrolysis. NFC and CNC are used for several reasons including the mechanical properties, the extended surface area and the low toxicity. Here we present some potential applications of nano-sized cellulose in the fields of wound healing, bone-cartilage regeneration, dental application and different human diseases including cancer. To witness the close proximity of nano-sized cellulose to the practical biomedical use, examples of recent clinical trials are also reported. Altogether, the described examples strongly support the enormous application potential of nano-sized cellulose in the biomedical field.
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Over the past ten years, the global biopharmaceutical market has remarkably grown, with ten over the top twenty worldwide high performance medical treatment sales being biologics. Thus, biotech R&D (research and development) sector is becoming a key leading branch, with expanding revenues. Biotechnology offers considerable advantages compared to traditional therapeutic approaches, such as reducing side effects, specific treatments, higher patient compliance and therefore more effective treatments leading to lower healthcare costs. Within this sector, smart nanotechnology and colloidal self-assembling systems represent pivotal tools able to modulate the delivery of therapeutics. A comprehensive understanding of the processes involved in the self-assembly of the colloidal structures discussed therein is essential for the development of relevant biomedical applications. In this review we report the most promising and best performing platforms for specific classes of bioactive molecules and related target, spanning from siRNAs, gene/plasmids, proteins/growth factors, small synthetic therapeutics and bioimaging probes.
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Sistemas de Liberación de Medicamentos/métodos , Hidrogeles/química , Terapia Molecular Dirigida , Nanopartículas/química , Nanotecnología/métodos , Animales , Anticuerpos/farmacología , Diagnóstico por Imagen/métodos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Liposomas/química , Liposomas/metabolismo , Ratones , Sondas Moleculares/síntesis química , Nanopartículas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , TransgenesRESUMEN
Viscosupplementation is a therapeutic approach for osteoarthritis treatment, where the synovial fluid, the natural lubricant of the joints, is replaced by viscoelastic solutions with rheological properties comparable or better than the starting material. This study presents the development of an innovative platform for viscosupplementation, based on the optimization of polysaccharide-based colloidal hydrogel, aiming to reduce on-site enzyme degradation and enhance the possibility of hyaluronic acid substitution with alternative biomaterials. Catanionic vesicles are proposed as physical crosslinker that can guarantee the formation of a 'soft', tunable network, offering a dual-therapeutic approach: on the mechanical relief perspective, as well as on the drug/gene delivery strategy. This research focuses on the fabrication and optimization of colloidal networks, driven by the synergistic interaction among catanionic vesicles and cationic modified cellulose polymers. This study tests the hypothesis that cellulose-like polymers can be arranged into functional matrix, mimicking the mechanical properties of healthy synovial fluids.
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Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Cartílago Articular/efectos de los fármacos , Elasticidad , Hidrogeles/química , Polisacáridos/química , Polisacáridos/farmacología , Cartílago Articular/metabolismo , Líquido Sinovial/metabolismo , ViscosidadRESUMEN
INTRODUCTION: hepatocellular carcinoma (hcc) is the predominant form of primary liver cancer and the second leading cause of cancer-associated mortality worldwide. available therapies for hcc have limited efficacy due to often late diagnosis and the general resistance of hcc to anti-cancer agents; therefore, the development of novel therapeutics is urgently required. small-interfering rna (sirna) molecules are short, double-stranded rnas that specifically recognize and bind the mrna of a target gene to inhibit gene expression. despite the great therapeutic potential of sirnas towards many human tumors including hcc, their use is limited by suboptimal delivery. Areas covered: In this review, we outline the current data regarding the therapeutic potential of siRNAs in HCC and describe the development of effective siRNA delivery systems. We detail the key problems associated with siRNA delivery and discuss the possible solutions. Finally, we provide examples of the various siRNA delivery strategies that have been employed in animal models of HCC and in human patients enrolled in clinical trials. Expert opinion: Despite the existing difficulties in siRNA delivery for HCC, the increasing scientific attention and breakthrough studies in this field is facilitating the design of novel and efficient technical solutions that may soon find practical applications.
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Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/genética , Línea Celular , Humanos , Neoplasias Hepáticas/genéticaRESUMEN
Eukaryotic elongation factor 1A (eEF1A), a protein involved in protein synthesis, has two major isoforms, eEF1A1 and eEF1A2. Despite the evidences of their involvement in hepatocellular carcinoma (HCC), the quantitative contribution of each of the two isoforms to the disease is unknown. We depleted the two isoforms by means of siRNAs and studied the effects in three different HCC cell lines. Particular care was dedicated to select siRNAs able to target each of the two isoform without affecting the other one. This is not a trivial aspect due to the high sequence homology between eEF1A1 and eEF1A2. The selected siRNAs can specifically deplete either eEF1A1 or eEF1A2. This, in turn, results in an impairment of cell vitality, growth and arrest in the G1/G0 phase of the cell cycle. Notably, these effects are quantitatively superior following eEF1A1 than eEF1A2 depletion. Moreover, functional tests revealed that the G1/G0 block induced by eEF1A1 depletion depends on the down-regulation of the transcription factor E2F1, a known player in HCC. In conclusion, our data indicate that the independent targeting of the two eEF1A isoforms is effective in reducing HCC cell growth and that eEF1A1 depletion may result in a more evident effect.
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Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , ARN Interferente Pequeño/administración & dosificación , Línea Celular Tumoral , Humanos , Liposomas/química , Isoformas de ProteínasRESUMEN
The lung alveoli slowly self-renew pneumocytes, but their facultative regeneration capacity is rapidly efficient after an injury, so fibrosis infrequently occurs. We recently observed Keratin 14 (KRT14) expression during diffuse alveolar damage (DAD), but not in controls. We wonder if KRT14 may be a marker of pneumocyte transition from quiescence to regeneration. Quantitative PCR and Western blot analyses highlighted the presence of KRT14 (mRNA and protein) only in human lung samples with DAD or interstitial lung disease (ILD). In the exponentially growing cell lines A549 and H441, the mRNA and protein levels of KRT14 peaked at day one after cell seeding and decreased at day two, opposite to what observed for the proliferation marker E2F1. The inverse relation of KRT14 versus E2F1 expression holds true also for other proliferative markers, such as cyclin E1 and cyclin D1. Of interest, we also found that E2F1 silencing caused cell cycle arrest and increased KRT14 expression, whilst E2F1 stimulation induced cell cycle progression and decreased KRT14. KRT14 also increased in proliferative pneumocytes (HPAEpiC) just before transdifferentiation. Overall, our results suggest that KRT14 is a viable biomarker of pneumocyte activation, and repair/regeneration. The involvement of KRT14 in regenerative process may suggest a novel pharmaceutical target to accelerate lung repair.
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Queratina-14/genética , Enfermedades Pulmonares Intersticiales/patología , ARN Mensajero/metabolismo , Células A549 , Adolescente , Anciano , Anciano de 80 o más Años , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Puntos de Control del Ciclo Celular , Proliferación Celular , Transdiferenciación Celular , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Femenino , Humanos , Queratina-14/antagonistas & inhibidores , Queratina-14/metabolismo , Enfermedades Pulmonares Intersticiales/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , TranscriptomaRESUMEN
The limited efficacy of available treatments for hepatocellular carcinoma (HCC) requires the development of novel therapeutic approaches. We synthesized a novel cationic polymer based on α,ß-poly-(N-2-hydroxyethyl)-d,L-aspartamide (PHEA) for drug delivery to HCC cells. The copolymer was synthesized by subsequent derivatization of PHEA with diethylene triamine (DETA) and with a polyethylene glycol (PEG) derivative bearing galactose (GAL) molecules, obtaining the cationic derivative PHEA-DETA-PEG-GAL. PHEA-DETA-PEG-GAL has suitable chemical-physical characteristics for a potential systemic use and can effectively deliver a siRNA (siE2F1) targeted against the transcription factor E2F1, a gene product involved in HCC. The presence of GAL residues in the polyplexes allows the targeting of HCC cells that express the asialo-glycoprotein receptor (ASGP-R). In these cells, but not in ASGP-R non-expressing cells, PHEA-DETA-PEG-GAL/siE2F1 polyplexes induce the reduction of the mRNA and protein levels of E2F1 and of E2F1-regulated genes, all involved in the promotion of the G1/S phase transition. This results in a decrease of cell proliferation with a G1/G0 phase cells accumulation. Notably, removal of GAL residue almost completely abrogates the targeting capacity of the developed polyplexes. In conclusion, the generated polyplexes demonstrate the potential to effectively contributing to the development of novel anti-HCC therapeutic approaches via a siRNA-targeted delivery.