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Plant-derived extracellular vesicles (EVs) have been recognized as important mediators of intercellular communication able to transfer active biomolecules across the plant and animal kingdoms. EVs have demonstrated an impressive array of biological activities, displaying preventive and therapeutic potential in mitigating various pathological processes. Indeed, the simplicity of delivering exogenous and endogenous bioactive molecules to mammalian cells with their low cytotoxicity makes EVs suitable agents for new therapeutic strategies for a variety of pathologies. In this study, EVs were isolated from Opuntia ficus-indica fruit (OFI-EVs) and characterized by particle size distribution, concentration, and bioactive molecule composition. OFI-EVs had no obvious toxicity and demonstrated a protective role in the inflammatory process and oxidative stress in vitro model of chronic skin wounds. The results demonstrated that pretreatment with OFI-EVs decreased the activity and gene expression of pro-inflammatory cytokines (IL-6, IL-8, and TNF-α) in the LPS-stimulated human leukemia monocytic cell line (THP-1). Furthermore, OFI-EVs promote the migration of human dermal fibroblasts (HDFs), speeding up the normal wound healing processes. This study sheds light, for the first time, on the role of OFI-EVs in modulating important biological processes such as inflammation and oxidation, thereby identifying EVs as potential candidates for healing chronic cutaneous wounds.
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Vesículas Extracelulares , Fibroblastos , Frutas , Opuntia , Cicatrización de Heridas , Opuntia/química , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Cicatrización de Heridas/efectos de los fármacos , Frutas/química , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Citocinas/metabolismo , Movimiento Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Células THP-1RESUMEN
The mucosal-dominant variant of pemphigus vulgaris (MPV) is an autoimmune disease characterized by oral mucosal blistering and circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3), resulting in life-threatening bullae and erosion formation. Recently, microRNAs (miRNAs) have emerged as promising players in the diagnosis and prognosis of several pathological states. For the first time, we have identified a different expression profile of miRNAs isolated from plasma-derived exosomes (P-EVs) of MPV patients positive for antibodies against Dsg3 (Dsg3-positive) compared to healthy controls. Moreover, a dysregulated miRNA profile was confirmed in MPV tissue biopsies. In particular, a strong downregulation of the miR-148a-3p expression level in P-EVs of MPV patients compared to healthy controls was demonstrated. Bioinformatics prediction analysis identifies metalloproteinase-7 (MMP7) as a potential miR-148a-3p target. An in vitro acantholysis model revealed that the miR-148a-3p expression level was dramatically downregulated after treatment with Dsg3 autoantibodies, with a concomitant increase in MMP7 expression. The increased expression of MMP7 leads to the disruption of intercellular and/or extracellular matrix adhesion in an in vitro cellular model of MPV, with subsequent cell dissociation. Overexpression of miR-148a-3p prevented cell dissociation and regressed MMP7 upregulation. Our findings suggest a pivotal role of P-EV cargo in regulating molecular mechanisms involved in MPV pathogenesis and indicate them as potential MPV therapeutic targets.
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MicroARNs , Pénfigo , Humanos , Pénfigo/genética , Pénfigo/diagnóstico , Regulación hacia Abajo/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Desmogleína 3/genética , Desmogleína 3/metabolismo , Autoanticuerpos , MicroARNs/genética , MicroARNs/metabolismo , Vesícula , Mucosa Bucal/metabolismoRESUMEN
The management of periodontitis remains a vital clinical challenge due to the interplay between the microorganisms of the dental biofilm and the host inflammatory response leading to a degenerative process in the surrounding tissues. Quercetin (QUE), a natural flavonol found in many foods, including apples, onions and tea, has exhibited prolonged and strong antibiofilm and anti-inflammatory effects both in vitro and in vivo. However, its clinical application is limited by its poor stability and water solubility, as well as its low bioavailability. Thus, in the present study, electrospun polylactic acid (PLA) nanofibers loaded with different amounts (5−10% w/w) of QUE were produced to rapidly respond to the acidic microenvironment typical of periodontal pockets during periodontal disease. This strategy demonstrated that PLA-QUE membranes can act as a drug reservoir releasing high QUE concentrations in the presence of oral bacterial infection (pH < 5.5), and thus limiting Pseudomonas aeruginosa PAO1 and Streptococcus mutans biofilm maturation. In addition, released QUE exerts antioxidant and anti-inflammatory effects on P. gingivalis Lipopolysaccharide (LPS)-stimulated human gingival fibroblast (HGFs). The reported results confirmed that PLA-QUE membranes could inhibit subgingival biofilm maturation while reducing interleukin release, thereby limiting host inflammatory response. Overall, this study provided an effective pH-sensitive drug delivery system as a promising strategy for treating periodontitis.
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Nanofibras , Periodontitis , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Humanos , Nanofibras/química , Periodontitis/tratamiento farmacológico , Poliésteres/química , Quercetina/químicaRESUMEN
Curcumin extracted from the rhizome of Curcuma Longa has been used in therapeutic preparations for centuries in different parts of the world. However, its bioactivity is limited by chemical instability, water insolubility, low bioavailability, and extensive metabolism. In this study, the coaxial electrospinning technique was used to produce both poly (ε-caprolactone) (PCL)-curcumin and core-shell nanofibers composed of PCL and curcumin in the core and poly (lactic acid) (PLA) in the shell. Morphology and physical properties, as well as the release of curcumin were studied and compared with neat PCL, showing the formation of randomly oriented, defect-free cylindrical fibers with a narrow distribution of the dimensions. The antibacterial and antibiofilm potential, including the capacity to interfere with the quorum-sensing mechanism, was evaluated on Pseudomonas aeruginosa PAO1, and Streptococcus mutans, two opportunistic pathogenic bacteria frequently associated with infections. The reported results demonstrated the ability of the Curcumin-loading membranes to inhibit both PAO1 and S. mutans biofilm growth and activity, thus representing a promising solution for the prevention of biofilm-associated infections. Moreover, the high biocompatibility and the ability to control the oxidative stress of damaged tissue, make the synthesized membranes useful as scaffolds in tissue engineering regeneration, helping to accelerate the healing process.
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Antiinfecciosos/farmacología , Biopelículas , Curcumina/farmacología , Infecciones/microbiología , Nanofibras/química , Ingeniería de Tejidos , Biopelículas/efectos de los fármacos , Compuestos de Bifenilo/química , Muerte Celular/efectos de los fármacos , Línea Celular , Liberación de Fármacos , Depuradores de Radicales Libres/farmacología , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Picratos/química , Poliésteres/química , Percepción de Quorum/efectos de los fármacos , TermogravimetríaRESUMEN
The interest in dietary polyphenols in recent years has greatly increased due to their antioxidant bioactivity with preventive properties against chronic diseases. Polyphenols, by modulating different cellular functions, play an important role in neuroprotection and are able to neutralize the effects of oxidative stress, inflammation, and apoptosis. Interestingly, all these mechanisms are involved in neurodegeneration. Although polyphenols display differences in their effectiveness due to interindividual variability, recent studies indicated that bioactive polyphenols in food and beverages promote health and prevent age-related cognitive decline. Polyphenols have a poor bioavailability and their digestion by gut microbiota produces active metabolites. In fact, dietary bioactive polyphenols need to be modified by microbiota present in the intestine before being absorbed, and to exert health preventive effects by interacting with cellular signalling pathways. This literature review includes an evaluation of the literature in English up to December 2019 in PubMed and Web of Science databases. A total of 307 studies, consisting of research reports, review articles and articles were examined and 146 were included. The review highlights the role of bioactive polyphenols in neurodegeneration, with a particular emphasis on the cellular and molecular mechanisms that are modulated by polyphenols involved in protection from oxidative stress and apoptosis prevention.
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Enfermedades Neurodegenerativas/tratamiento farmacológico , Polifenoles/farmacología , Polifenoles/uso terapéutico , Animales , Antioxidantes/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Neuroprotección/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.
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Carcinogénesis/metabolismo , Movimiento Celular , Proliferación Celular , Portadores de Fármacos , MicroARNs , Nanopartículas/química , Neoplasias de la Próstata/metabolismo , Células CACO-2 , Carcinogénesis/patología , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Humanos , Células MCF-7 , Masculino , MicroARNs/química , MicroARNs/farmacología , Células PC-3 , Prohibitinas , Neoplasias de la Próstata/patologíaRESUMEN
Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.
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Enfermedad de Huntington/tratamiento farmacológico , Metilhidrazinas/uso terapéutico , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Animales , Animales Modificados Genéticamente , Muerte Celular/efectos de los fármacos , Línea Celular , Medio de Cultivo Libre de Suero , Modelos Animales de Enfermedad , Drosophila , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/patología , Metilhidrazinas/farmacología , Modelos Biológicos , Mutación/genética , Agregado de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Análisis de Supervivencia , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Oral diseases encompassing conditions such as oral cancer, periodontitis, and endodontic infections pose significant challenges due to the oral cavity's susceptibility to pathogenic bacteria and infectious agents. Saliva, a key component of the oral environment, can compromise drug efficacy during oral disease treatment by diluting drug formulations and reducing drug-site interactions. Thus, it is imperative to develop effective drug delivery methods. Stimuli-responsive nanocomposite hydrogels offer a promising solution by adapting to changes in environmental conditions during disease states, thereby enabling targeted drug delivery. These smart drug delivery systems have the potential to enhance drug efficacy, minimize adverse reactions, reduce administration frequency, and improve patient compliance, thus facilitating a faster recovery. This review explores various types of stimuli-responsive nanocomposite hydrogels tailored for smart drug delivery, with a specific focus on their applications in managing oral diseases.
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Atopic dermatitis (AD) is a common disease-causing skin inflammation, redness, and irritation, which can eventually result in infection that drastically impacts patient quality of life. Resveratrol (Res) is a natural phytochemical famed for its excellent anti-inflammatory and antioxidant activities. However, it is poorly bioavailable. Thus, a drug delivery system is needed to enhance in vivo bioactivity. Herein, we report the preparation of hyaluronic acid (HA) hydrogels containing resveratrol-loaded chitosan (CS) nanoparticles, their physicochemical analysis, and their potential therapeutic effects in the treatment of AD. Positively charged CS nanoparticles prepared by tripolyphosphate (TPP) gelation showed sizes ranging from 120 to around 500 nm and Res encapsulation efficiency as high as 80%. Embedding the nanoparticles in HA retarded their hydrolytic degradation and also slowed resveratrol release. Resveratrol released from nanoparticle-loaded hydrogel counteracted the oxidative damage induced by ROS generation in TNF-α/INF-γ-treated human keratinocytes (HaCaT) used as an AD in vitro model. Moreover, pre-treatment with Res@gel reduced secretion and gene expression of proinflammatory cytokines in HaCaT cells. The physicochemical analysis and in vitro assay confirmed that the formulated hydrogel could be considered an efficient and sustained resveratrol delivery vector in AD treatment.
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OBJECTIVE: Dentin-pulp complex is object of interest in the regenerative endodontic field as well as the natural function of human dental pulp stem cells (hDPSCs) that may differentiate into specific cells able to repair and/or regenerate both hard and soft dental structures. The aim of the present study was to evaluate the capacity of hDPSCs to differentiate in odontoblastic-like cells by evaluating the expression of specific odontogenic-related genes and to prove the ability of treatment with calcium-based materials such as calcium carbonate (CaCO3), calcium hydroxide (Ca(OH)2), and mineral trioxide aggregate (MTA). METHODS: hDPSCs were obtained and isolated from a third molar of a young patient. Odontogenic-related gene expression was assessed unti1 28 days of culture as well as alkaline phosphatase activity (ALP). hDPSCs were cultured in odontoblastic-induction medium used as control, and in presence of different concentrations of CaCO3, Ca(OH)2, and MTA. RESULTS: The results demonstrated an upregulation in odontoblastic cell-related genes, in particular of the early differentiation marker known as matrix extracellular phosphoglycoprotein (MEPE), as well as increased ALP activity and the presence of calcium deposits, mainly by stimulation with calcium derivatives. In this regard, treatment of pulp tissue with CaCO3, Ca(OH)2 and even better with MTA seemed to be effective for dentinogenesis. SIGNIFICANCE: The ease of isolation of hDPSCs from discarded or extracted teeth offers a promising source of autologous cells that may be applied for regenerative purpose in combination with selected bioactive materials. However, further investigations should be conducted to confirm the obtained results.
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Calcio , Pulpa Dental , Humanos , Diferenciación Celular , Odontoblastos , Dentina , Expresión Génica , Células CultivadasRESUMEN
Diabetic wound infections (DWI) represent one of the most costly and disruptive complications in diabetic mellitus. The hyperglycemic state induces a persistent inflammation with immunological and biochemical impairments that promotes delayed wound healing processes and wound infection that often results in extended hospitalization and limb amputations. Currently, the available therapeutic options for the management of DWI are excruciating and expensive. Hence, it is essential to develop and improve DWI-specific therapies able to intervene on multiple fronts. Quercetin (QUE) exhibits excellent anti-inflammatory, antioxidant, antimicrobial and wound healing properties, which makes it a promising molecule for the management of diabetic wounds. In the present study, Poly-lactic acid/poly(vinylpyrrolidone) (PP) co-electrospun fibers loaded with QUE were developed. The results demonstrated a bimodal diameter distribution with contact angle starting from 120°/127° and go to 0° in less than 5 s indicating the hydrophilic nature of fabricated samples. The release QUE kinetics, analyzed in simulated wound fluid (SWF), revealed a strong initial burst release, followed by a constant and continuous QUE release. Moreover, QUE-loaded membranes present excellent antibiofilm and anti-inflammatory capacity and significantly reduce the gene expression of M1 markers tumor necrosis factor (TNF)-α, and IL-1ß in differentiated macrophages. In conclusion, the results suggested that the prepared mats loaded with QUE could be a hopeful drug-delivery system for the effective treatment of diabetic wound infections.
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Although osteoarthritis (OA) is a chronic inflammatory degenerative disease affecting millions of people worldwide, the current therapies are limited to palliative care and do not eliminate the necessity of surgical intervention in the most severe cases. Several dietary and nutraceutical factors, such as hydroxytyrosol (Hyt), have demonstrated beneficial effects in the prevention or treatment of OA both in vitro and in animal models. However, the therapeutic application of Hyt is limited due to its poor bioavailability following oral administration. In the present study, a localized drug delivery platform containing a combination of Hyt-loading chitosan nanoparticles (Hyt-NPs) and in situ forming hydrogel have been developed to obtain the benefits of both hydrogels and nanoparticles. This thermosensitive formulation, based on Pluronic F-127 (F-127), hyaluronic acid (HA) and Hyt-NPs (called Hyt@tgel) presents the unique ability to be injected in a minimally invasive way into a target region as a freely flowing solution at room temperature forming a gel at body temperature. The Hyt@tgel system showed reduced oxidative and inflammatory effects in the chondrocyte cellular model as well as a reduction in senescent cells after induction with H2O2. In addition, Hyt@tgel influenced chondrocytes gene expression under pathological state maintaining their metabolic activity and limiting the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features. Hence, it can be concluded that the formulated hydrogel injection could be proposed for the efficient and sustained Hyt delivery for OA treatment. The next step would be the extraction of "added-value" bioactive polyphenols from by-products of the olive industry, in order to develop a green delivery system able not only to enhance the human wellbeing but also to promote a sustainable environment.
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The beneficial effects of the Mediterranean diet (MedDiet), the most widely followed healthy diet in the world, are principally due to the presence in the foods of secondary metabolites, mainly polyphenols, whose healthy characteristics are widely recognized. However, one of the biggest problems associated with the consumption of polyphenols as nutraceutical adjuvant concerns their bioavailability. During the last decades, different nanotechnological approaches have been developed to enhance polyphenol bioavailability, avoiding the metabolic modifications that lead to low absorption, and improving their retention time inside the organisms. This review focuses on the most recent findings regarding the encapsulation and delivery of the bioactive molecules present in the foods daily consumed in the MedDiet such as olive oil, wine, nuts, spice, and herbs. In addition, the possibility of recovering the polyphenols from food waste was also explored, taking into account the increased market demand of functional foods and the necessity to obtain valuable biomolecules at low cost and in high quantity. This circular economy strategy, therefore, represents an excellent approach to respond to both the growing demand of consumers for the maintenance of human wellness and the economic and ecological exigencies of our society.
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To date, the implant-associated infections represent a worldwide challenge for the recently reported bacterial drug resistance that can lead to the inefficacy or low efficacy of conventional antibiotic therapies. Plant polyphenolic compounds, including resveratrol (RSV), are increasingly gaining consensus as valid and effective alternatives to antibiotics limiting antibiotic resistance. In this study, electrospun polylactic acid (PLA) membranes loaded with different concentrations of RSV are synthesized and characterized in their chemical, morphological, and release features. The obtained data show that the RSV release rate from the PLA-membranes is remarkably higher in acidic conditions than at neutral pH. In addition, a change in pH from neutral to slightly acidic triggers a significant increase in the RSV release. This behavior indicates that the PLA-RSV membranes can act as drug reservoir when the environmental pH is neutral, starting to release the bioactive molecules when the pH decreases, as in presence of oral bacterial infection. Indeed, our results demonstrate that PLA-RSV2 displays a significant antibacterial and antibiofilm activity against two bacterial strains, Pseudomonas aeruginosa PAO1, and Streptococcus mutans, responsible for both acute and chronic infections in humans, thus representing a promising solution for the prevention of the implant-associated infections.
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The introduction of druggable targets has significantly improved the outcomes of non-small cell lung cancer patients (NSCLC). EML4-ALK translocation represents 4-6% of the druggable alterations in NSCLC. With the approval of Crizotinib, first discovered drug for the EML4-ALK translocation, on first line treatment for patients with detected mutation meant a complete change on the treatment landscape. The current standard method for EML4-ALK identification is immunohistochemistry or FISH in a tumor biopsy. However, a big number of NSCLC patients have not tissue available for analysis and others are not suitable for biopsy due to their physical condition or the location of the tumor. Liquid biopsy seems the best alternative for identification in these patients that have no tissue available. Circulating free RNA has not been validated for the identification of this mutation. As a complementary tool, exosomes might represent a good tool for predictive biomarkers study, and due to their stability, they preserve the genetic material contained in them. Our group has described for the first time the translocation EML4-ALK in RNA isolated from exosomes derived from NSCLC patients using next generation sequencing.
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Metabolic flexibility describes the ability of cells to respond or adapt its metabolism to support and enable rapid proliferation, continuous growth, and survival in hostile conditions. This dynamic character of the cellular metabolic network appears enhanced in cancer cells, in order to increase the adaptive phenotype and to maintain both viability and uncontrolled proliferation. Cancer cells can reprogram their metabolism to satisfy the energy as well as the biosynthetic intermediate request and to preserve their integrity from the harsh and hypoxic environment. Although several studies now recognize these reprogrammed activities as hallmarks of cancer, it remains unclear which are the pathways involved in regulating metabolic plasticity. Recent findings have suggested that carnitine system (CS) could be considered as a gridlock to finely trigger the metabolic flexibility of cancer cells. Indeed, the components of this system are involved in the bi-directional transport of acyl moieties from cytosol to mitochondria and vice versa, thus playing a fundamental role in tuning the switch between the glucose and fatty acid metabolism. Therefore, the CS regulation, at both enzymatic and epigenetic levels, plays a pivotal role in tumors, suggesting new druggable pathways for prevention and treatment of human cancer.
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Carnitina/metabolismo , Epigénesis Genética , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transporte Biológico , Carnitina Aciltransferasas/genética , Carnitina Aciltransferasas/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Metabolismo Energético/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolismo de los Lípidos , MicroARNs/genética , MicroARNs/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Lung cancer is a highly lethal disease. Targeted therapies have been developed in last years, however survival rates are not improving due to the delay in the diagnosis, making biomarkers one of the most interesting fields of study in cancer. Liquid biopsy has raised as an alternative to tissue biopsy due to improvements in analytical techniques for circulating tumor cells (CTCs), cell free DNA and exosomes. Among all, exosomes have raised as one of the most promising tools to understand the tumor due to their stability in the blood and their similarity to the cells of origin. In the last years, different alterations have been described inside the exosomes derived from non-small cell lung cancer (NSCLC) cells mirroring the processes inside these tumoral cells, such as EGFR mutation, translocations or microRNA (miRNA) deregulation. All these studies have opened the window to a new world of possibilities in the study of tumor biomarkers.
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Understanding molecular mechanisms involved in melanoma resistance to drugs is a big challenge. Experimental evidences suggested a correlation between mutational status in B-RAF and melanoma cell susceptibility to drugs, such as paclitaxel, doxorubicin and temozolomide, which generate an accumulation of hydrogen peroxide (H2O2) in the cells. We investigated the survival phenotype and the protein level of c-myc, a B-RAF target molecule, in melanoma cells, carrying a different mutational status in B-RAF, upon paclitaxel, doxorubicin and H2O2 treatment. For the first time, we reported c-myc modulation is critical for melanoma drug response. It appeared drug-specific and post-transcriptionally driven through PP2A; in correlation, cell pre-treatment with okadaic acid (OA), a specific PP2A inhibitor, as well as PP2A silencing of melanoma cells, was able to increase melanoma cell drug-sensitivity and c-myc protein level. This is relevant for designing efficacious therapeutic strategies in melanoma.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Melanoma/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias Cutáneas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Melanoma/tratamiento farmacológico , Paclitaxel/farmacología , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias Cutáneas/tratamiento farmacológico , TemozolomidaRESUMEN
We performed a comparative study between two human metastatic melanoma cell lines (A375 and 526), and melanocytes (FOM78) by gene expression profiling and pathway analysis, using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) software. Genes involved in Ran signaling were significantly over-represented (p ≤ 0.001) and up-regulated in melanoma cells. A melanoma-associated molecular pathway was identified, where Ran, Aurora Kinase A (AurkA) and TERT were up-regulated, while c-myc and PTEN were down-regulated. A consistent high Ran and AurkA gene expression was detected in about 48% and 53%, respectively, of 113 tissue samples from metastatic melanoma patients. AurkA down-regulation was observed in melanoma cells, by Ran knockdown, suggesting AurkA protein is a Ran downstream target. Furthermore, AurkA inhibition, by exposure of melanoma cells to MLN8054, a specific AurKA inhibitor, induced apoptosis in both melanoma cell lines and molecular alterations in the IPA-identified molecular pathway. These alterations differed between cell lines, with an up-regulation of c-myc protein level observed in 526 cells and a slight reduction seen in A375 cells. Moreover, Ran silencing did not affect the A375 invasive capability, while it was enhanced in 526 cells, suggesting that Ran knockdown, by AurkA down-regulation, resulted in a Ran-independent enhanced melanoma cell invasion. Finally, AurK A inhibition induced a PTEN up-regulation and its action was independent of B-RAF mutational status. These findings provide insights relevant for the development of novel therapeutic strategies as well as for a better understanding of mechanisms underlying therapy resistance in melanoma.
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Melanoma/genética , Melanoma/metabolismo , Proteína de Unión al GTP ran/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Melanoma/patología , Transducción de Señal , Transfección , Proteína de Unión al GTP ran/genéticaRESUMEN
BACKGROUND: Down syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown. METHODS: To better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade. RESULTS: We show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes. CONCLUSIONS: The present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS.