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Wound healing is a challenging clinical problem and efficient wound management is essential to prevent infection. This is best done by utilizing biocompatible materials in order to complete the healing in a rapid manner, with functional and esthetic outcomes. In this context, the zein protein fulfills the criteria of the ideal wound dressing which include non-toxicity and non-inflammatory stimulation. Zein gels containing rutin were prepared without any chemical refinement or addition of gelling agents in order to obtain a natural formulation characterized by antioxidant and anti-inflammatory properties to be proposed for the treatment of burns and sores. In vitro scratch assay showed that the proposed gel formulations promoted cell migration and a rapid gap closure within 24 h (~90 %). In addition, the in vivo activities of rutin-loaded zein gel showed a greater therapeutic efficacy in Wistar rats, with a decrease of the wound area of about 90 % at day 10 with respect to the free form of the bioactive and to DuoDERM®. The evaluation of various markers (TNF-α, IL-1ß, IL-6, IL-10) confirmed the anti-inflammatory effect of the proposed formulation. The results illustrate the feasibility of exploiting the peculiar features of rutin-loaded zein gels for wound-healing purposes.
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Materiales Biocompatibles , Geles , Ratas Wistar , Rutina , Cicatrización de Heridas , Zeína , Rutina/química , Rutina/farmacología , Zeína/química , Cicatrización de Heridas/efectos de los fármacos , Animales , Geles/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Ratas , Antiinflamatorios/farmacología , Antiinflamatorios/química , Masculino , Antioxidantes/farmacología , Antioxidantes/química , Tecnología Química Verde , Movimiento Celular/efectos de los fármacos , Humanos , Citocinas/metabolismoRESUMEN
BACKGROUND: Temporomandibular joint (TMJ) disorders, which affect millions of people worldwide, have multiple etiological factors that make an accurate diagnosis and effective treatments difficult. As a consequence, the gold standard diagnostic criteria for TMJ disorders remain elusive and often depend on subjective decisions. AIM: In this context, the lack of a non-invasive quantitative methodology capable of assessing the functional physiological state and, consequently, identifying risk indicators for the early diagnosis of TMJ disorders must be tackled and resolved. METHODOLOGY: In this work, we have studied the biomechanics and viscoelastic properties of the functional masticatory system by a non-invasive approach involving 52 healthy subjects, analysed by statistical-physics analysis applied to myotonic measurements on specific points of the masticatory system designing a TMJ network composed of 17 nodes and 20 links. RESULTS: We find that the muscle tone and viscoelasticity of a specific cycle linking frontal, temporal, and mandibular nodes of the network play a prominent role in the physiological functionality of the system. At the same time, the functional state is characterised by a landscape of nearly degenerated levels of elasticity in all links of the network, making this parameter critically distributed and deviating from normal behaviour. CONCLUSIONS: Time evolution and dynamic correlations between biomechanics and viscoelastic parameters measured on the different cycles of the network provide a quantitative framework associated with the functional state of the masticatory system. Our results are expected to contribute to enriching the taxonomy of this system, primarily based on clinical observations, patient symptoms, and expert consensus.
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In the last few decades, several natural and synthetic polymers have been used as starting material for the development of innovative polymeric nanoparticles able to encapsulate biologically active substances and to modulate their biopharmaceutical features and/or therapeutic efficacy. This investigation focused on the comparison of the physico-chemical properties of nanosystems made up of two of the most successfully used biodegradable biomaterials, namely poly(lactic-co-glycolic acid) (PLGA) and zein, belonging to the synthetic and natural family of polymers, respectively. Rutin, a polyphenolic bioflavonoid characterized by peculiar antioxidant properties, was chosen as the model drug to be encapsulated in the polymeric systems. The results demonstrated a greater ability of zein-based nanosystems to effectively retain the active compound with respect to the PLGA particles. The integration of rutin in the protein matrix favored a controlled drug leakage, and was influenced by the surfactant used to stabilize the formulation. Moreover, rutin-loaded zein nanoparticles showed significant in vitro antioxidant activity, evidencing a synergistic action between the intrinsic antioxidant activity of the protein and the pharmacological properties of the active compound. Finally, the intracellular localization of the zein nanosystems was demonstrated through confocal laser scanning microscopy.
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Nanopartículas , Zeína , Portadores de Fármacos , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros , RutinaRESUMEN
Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)-dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.
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The development of innovative strategies for the efficacious treatment of inflammatory bowel diseases (IBD) still remains a goal for pharmaceutical research. Targeting the lower section of the intestine is the main aim of therapy because it is the compartment primarily affected by IBDs. Mesalazine was microencapsulated in chitosan particles in order to modulate its unfavorable pharmacokinetic profile exploiting the bioadhesive feature of the polysaccharide and increase the anti-inflammatory effect of the drug following its rectal administration in an in vivo model of induced IBD. The chitosan microparticles (1-4⯵m mean size) allowed efficient retention of the mesalazine and a prolonged drug release lasting up to 48â¯h. In vitro and in vivo experiments confirmed the significant mucoadhesion feature of the formulation by means of mucin assay and CLSM experiments and demonstrated its therapeutic efficacy at a drug concentration 2-fold lower than the commercial formulation Asamax® (13â¯mg/kg vs 26â¯mg/kg).
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Antiinflamatorios no Esteroideos/administración & dosificación , Quitosano/administración & dosificación , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Mesalamina/administración & dosificación , Microesferas , Administración Rectal , Animales , Antiinflamatorios no Esteroideos/química , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Quitosano/química , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Sulfato de Dextran/toxicidad , Humanos , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/metabolismo , Mesalamina/química , Ratones , Distribución Aleatoria , Resultado del TratamientoRESUMEN
Many reports indicate that the protective action of nutraceuticals in the Mediterranean diet, against metabolic and cardiovascular diseases, can be attributed to the action of polyphenolic components of extra-virgin olive oil (EVOO). Here, we evaluated the protective effects of oleacein, one of the most abundant secoiridoids in EVOO, on the damages/metabolic alterations caused by high-fat diet (HFD) in male C57BL/6JolaHsd mice. After 5 weeks of treatment with 20 mg/kg of oleacein, body weight, glycemia, insulinemia, serum lipids, and histologic examination of liver tissue indicated a protective action of oleacein against abdominal fat accumulation, weight gain, and liver steatosis, with improvement of insulin-dependent glucose and lipid metabolism. Both serum parameters and hepatic histologic examination were altered in mice fed with HFD. By contrast, in the animals that received oleacein, plasma glucose, cholesterol and triglyceride serum levels, and liver histology were similar to controls fed with normocaloric diet. In addition, protein levels of FAS, SREBP-1, and phospho-ERK in liver were positively modulated by oleacein, indicating an improvement in liver insulin sensitivity. In a group of obese mice, treatment with oleacein determined a light, but still significant reduction of the increase in body weight, mainly due to lesser liver steatosis enlargement, associated with reduced levels of SREBP-1 and phospho-ERK and lower levels of total serum cholesterol; in these animals, altered plasma glucose and triglyceride serum levels were not reverted by oleacein. These results indicate that HFD-related hepatic insulin resistance may be partially prevented by oral administration of oleacein, suggesting a protective role of this nutraceutical against diet-dependent metabolic alterations. Additional studies are necessary to check whether oleacein can be used as an adjuvant to improve insulin sensitivity in humans.
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Embora o que se entenda como infidelidade varie de acordo com o contexto sociocultural e o contrato estabelecido por cada casal, seu impacto na relação costuma ser significativo e desafiador. Neste artigo, serão abordadas possibilidades de intervenção junto a casais que buscam terapia em virtude da infidelidade conjugal. É importante considerar a infidelidade como um fenômeno complexo, relacionado a questões intrapsíquicas, relacionais, situacionais e socioculturais. O terapeuta deve abordar (a) o impacto inicial da descoberta da infidelidade; (b) a história da relação, de forma a contextualizar o acontecido; e (c) o seu futuro. Nesse processo, mostram-se úteis tanto sessões individuais como conjugais. O entendimento da infidelidade requer um olhar sistêmico, que consiga abranger a história do casal e da família, suas crenças e seus padrões relacionais. O terapeuta precisa ser capaz de criar um ambiente de compreensão mútua que possibilite as mudanças desejadas pelos cônjuges.(AU)
Although its meaning may differ according to specific socio-cultural contexts and to the established contract, infidelity always causes stress on couples' relationships and is very challenging to deal with. This article addresses possibilities of intervention when dealing with couples who seek therapy due to infidelity. It must be considered as a complex phenomenon, associated to intrapsychic, relational, situational and socio-cultural issues. Therapists must address: (a) the initial impact of the of infidelity revelation; (b) the history of the relationship, in order to contextualize what happened; and (c) what can be expected for the relationship's future. In this process, both individual and couples sessions are useful. Understanding infidelity requires a systemic approach, which should consider the history of the couple and of their family; their beliefs and their relational patterns. Therapists should be able to create an environment of mutual understanding thus contributing to the changes desired by the spouses.(AU)
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Humanos , Relaciones Extramatrimoniales/psicología , Terapia Conyugal/métodos , Matrimonio/psicología , Conflicto Familiar/psicologíaRESUMEN
Rutin was microencapsulated in a chitosan matrix using the spray-drying technique and the resulting system was investigated. High amounts of rutin were efficiently entrapped within polymeric microspheres, and these microparticles were characterized by a smooth surface and afforded a controlled release of the active compound. The anti-inflammatory activity of rutin-loaded microspheres was investigated in in vitro models of NCTC 2544 and C-28 cells treated with LPS by determining the levels of IL-1ß and IL-6. The rutin-loaded microspheres showed an increase of in vitro anti-inflammatory activity with respect to the free active compound. Confocal laser scanning microscopy demonstrated that massive intracellular uptake of the chitosan microspheres took place after a few hours of incubation and that the drug was localized in the cytosol compartment of the treated cells. The improved anti-inflammatory activity of the rutin-loaded microspheres was further confirmed by an in vivo model of carrageenan-induced paw edema.
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Antiinflamatorios , Quitosano , Portadores de Fármacos , Microesferas , Rutina , Antiinflamatorios/química , Antiinflamatorios/farmacología , Línea Celular , Quitosano/química , Quitosano/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Rutina/química , Rutina/farmacologíaRESUMEN
An increased incidence of obesity is registered worldwide, and its association with insulin resistance and type 2 diabetes is closely related with increased morbidity and mortality for cardiovascular diseases. A major clinical problem in the management of obesity is the non-adherence or low adherence of patients to a hypocaloric dietetic restriction. In this study, we evaluated in obese mice the effects of shifting from high-calorie foods to normal diet on insulin sensitivity. Male C57BL/6JOlaHsd mice (n = 20) were fed with high fat diet (HFD) for a 24-week period. Afterward, body weight, energy, and food intake were measured in all animals, together with parameters of insulin sensitivity by homeostatic model assessment of insulin resistance and plasma glucose levels in response to insulin administration. Moreover, in half of these mice, Glut4 mRNA levels were measured in muscle at the end of the high fat treatment, whereas the rest of the animals (n = 10) were shifted to normocaloric diet (NCD) for 10 weeks, after which the same analyses were carried out. A significant reduction of body weight was found after the transition from high to normal fat diet, and this decrease correlated well with an improvement in insulin sensitivity. In fact, we found a reduction in serum insulin levels and the recovery of insulin responsiveness in terms of glucose disposal measured by insulin tolerance test and Glut4 mRNA and protein expression. These results indicate that obesity-related insulin resistance may be rescued by shifting from HFD to NCD.
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BACKGROUND: Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs. METHODS: Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program. RESULTS: We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133(pos)CD45(neg) (5 ± 1) and (2 ± 1) and CK20(pos)CD45(neg) of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4(pos) of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded-CTCs (eCTCs). ECTCs organized in tumor sphere were CD45(neg)CD133(pos) while in adhesion were CXCR4(pos)CK20(pos). These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45(neg)CD133(pos)) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4(pos)CK20(pos)) have a low disease-free survival. CONCLUSION: On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic-risk personalized score thus improving disease management and reducing patient care cost.
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Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Neoplasias del Colon/patología , Células Neoplásicas Circulantes/patología , Células Madre Neoplásicas/patología , Medicina de Precisión , Medición de Riesgo , Adulto , Anciano , Animales , Adhesión Celular , Ciclo Celular , Proliferación Celular , Forma de la Célula , Supervivencia Celular , Citocinas/metabolismo , Femenino , Humanos , Masculino , Ratones SCID , Persona de Mediana Edad , Metástasis de la Neoplasia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries. Defective autophagy of lipid droplets (LDs) in hepatocytes, also known as lipophagy, has recently been identified as a possible pathophysiological mechanism of NAFLD. Experimental and epidemiological evidence suggests that dietary polyphenols may prevent NAFLD. To address this hypothesis and analyze the underlying mechanisms, we supplemented bergamot polyphenol fraction (BPF) to cafeteria (CAF) diet-fed rats, a good model for pediatric metabolic syndrome and NAFLD. BPF treatment (50 mg/kg/day supplemented with drinking water, 3 months) potently counteracted the pathogenic increase of serum triglycerides and had moderate effects on blood glucose and obesity in this animal model. Importantly, BPF strongly reduced hepatic steatosis as documented by a significant decrease in total lipid content (-41.3% ± 12% S.E.M.), ultrasound examination and histological analysis of liver sections. The morphometric analysis of oil-red stained sections confirmed a dramatic reduction in LDs parameters such as total LD area (48.5% ± 15% S.E.M.) in hepatocytes from CAF+BPF rats. BPF-treated livers showed increased levels of LC3 and Beclin 1 and reduction of SQSTM1/p62, suggesting autophagy stimulation. Consistent with BPF stimulation of lipophagy, higher levels of LC3II were found in the LD subcellular fractions of BPF-expose livers. This study demonstrates that the liver and its lipid metabolism are the main targets of bergamot flavonoids, supporting the concept that supplementation of BPF is an effective strategy to prevent NAFLD.
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Citrus/química , Suplementos Dietéticos , Modelos Animales de Enfermedad , Lipotrópicos/uso terapéutico , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Extractos Vegetales/uso terapéutico , Polifenoles/uso terapéutico , Animales , Fármacos Antiobesidad/uso terapéutico , Autofagia , Biomarcadores/sangre , Biomarcadores/metabolismo , Dieta Occidental/efectos adversos , Frutas/química , Humanos , Italia , Gotas Lipídicas/diagnóstico por imagen , Gotas Lipídicas/metabolismo , Gotas Lipídicas/patología , Hígado/diagnóstico por imagen , Hígado/metabolismo , Hígado/patología , Síndrome Metabólico/etiología , Síndrome Metabólico/fisiopatología , Proteínas Asociadas a Microtúbulos/agonistas , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Obesidad/fisiopatología , Obesidad/prevención & control , Distribución Aleatoria , Ratas Wistar , UltrasonografíaRESUMEN
This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array (HPLC-PDA) method for the simultaneous analysis, in mouse plasma, of eperisone hydrochloride and paracetamol by protein precipitation using zinc sulphate-methanol-acetonitrile. The analytes were resolved on a Gemini C18 column (4.6 mm × 250 mm; 5 µm particle size) using a gradient elution mode with a run time of 15 min, comprising re-equilibration, at 60°C (± 1°C). The method was validated over the concentration range from 0.5 to 25 µg/mL for eperisone hydrochloride and paracetamol, in mouse plasma. Ciprofloxacin was used as Internal Standard. Results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.5 µg/mL for eperisone hydrochloride and paracetamol, and matrix-matched standard curves showed a good linearity, up to 25 µg/mL with correlation coefficients (r(2))≥ 0.9891. In the entire analytical range the intra and inter-day precision (RSD%) values were ≤ 1.15% and ≤ 1.46% for eperisone hydrochloride, and ≤ 0.35% and ≤ 1.65% for paracetamol. For both analytes the intra and inter-day trueness (bias%) values ranged, respectively, from -5.33% to 4.00% and from -11.4% to -4.00%. The method was successfully tested in pharmacokinetic studies after oral administration in mouse. Furthermore, the application of this method results in a significant reduction in terms of animal number, dosage, and improvement in speed, rate of analysis, and quality of pharmacokinetic parameters related to serial blood sampling.
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Acetaminofén/sangre , Cromatografía Líquida de Alta Presión/métodos , Propiofenonas/sangre , Acetaminofén/administración & dosificación , Acetaminofén/farmacocinética , Administración Oral , Animales , Área Bajo la Curva , Calibración , Cromatografía Líquida de Alta Presión/instrumentación , Ratones , Ratones Endogámicos C57BL , Propiofenonas/administración & dosificación , Propiofenonas/farmacocinética , Estándares de Referencia , Distribución Tisular , Estudios de Validación como AsuntoRESUMEN
B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.
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Anticuerpos Antiidiotipos/inmunología , Inmunoterapia/métodos , Linfoma de Células B/inmunología , Péptidos/inmunología , Animales , Anticuerpos Antiidiotipos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Idiotipos de Inmunoglobulinas/inmunología , Linfoma de Células B/terapia , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Biblioteca de Péptidos , Péptidos/uso terapéutico , Tomografía de Emisión de Positrones , Receptores de Antígenos de Linfocitos B/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Serum and glucocorticoid regulated kinase 1 (Sgk1) is a serine-threonine kinase that is activated by serum, steroids, insulin, vasopressin, and interleukin 2 at the transcriptional and post-translational levels. Sgk1 is also important in transduction of growth factors and steroid-dependent survival signals and may have a role in the development of resistance to cancer chemotherapy. In the present paper, we demonstrate that Sgk1 activates MDM2-dependent p53 ubiquitylation. The results were obtained in RKO cells and other cell lines by Sgk1-specific RNA silencing and were corroborated in an original mouse model as well as in transiently and in stably transfected HeLa cells expressing wild-type or dominant negative Sgk1 mutant. Sgk1 contributes to cell survival, cell-cycle progression, and epithelial de-differentiation. We also show that the effects of Sgk1 on the clonogenic potential of different cancer cells depend on the expression of wild-type p53. Since transcription of Sgk1 is activated by p53, we propose a finely tuned feedback model where Sgk1 down-regulates the expression of p53 by enhancing its mono- and polyubiquitylation.
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Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Proteínas Inmediatas-Precoces , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor , Animales , Muerte Celular , Línea Celular , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Gemcitabine (GEM) is presently the standard option for the treatment of advanced pancreatic cancer (PC). We investigated the in vitro and in vivo antitumor potential of GEM-loaded PEGylated liposomes (L-GEM) as a novel agent for the treatment of PC. In vitro analysis of antitumor activity against human PC cell lines, BXPC-3 and PSN-1, showed a significant time- and dose-dependent reduction of cell viability following exposure to L-GEM as compared to free GEM [at 72 h, IC(50): 0.009 vs. 0.027 microM (P = 0.003) for BXPC-3 and 0.003 vs. 0.009 microM (P < 0.001) for PSN1, respectively]. Confocal laser scanning microscopy demonstrated an effective liposome/cell interaction and internalization process following 3-h cell exposure to L-GEM. The in vivo antitumor activity of L-GEM was investigated in a cohort of SCID mice bearing BxPC-3 or PSN-1 xenografts. Animals were i.p. treated with L-GEM (5 mg/kg), or a threefold increased dose of free GEM (15 mg/kg), or empty liposomes or vehicle, twice a week for 35 days. A significant higher inhibition of tumor growth in mice treated with L-GEM versus free GEM (P = 0.006 and P = 0.004 for BXPC-3 and PSN-1, respectively) or control groups (P = 0.0001), translated in a survival advantage of L-GEM treated animals versus other groups. Pharmacokinetic studies showed enhancement of systemic bioavailability of L-GEM (t (1/2) = 8 h) versus to GEM (t (1/2) = 1.5 h). Our findings demonstrate that L-GEM is an effective agent against PC and exerts higher antitumor activity as compared to free GEM with no appreciable increase in toxicity. These results provide the pre-clinical rational for L-GEM clinical development for the treatment of PC patients.
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Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapéutico , Portadores de Fármacos , Semivida , Humanos , Liposomas , Ratones , Ratones SCID , Microscopía Confocal , Polietilenglicoles/química , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The aim of present work was the evaluation of the effects on brain levels of nerve growth factor (NGF) and of its high-affinity tyrosine kinase A receptor (TrkA), induced in rats unilaterally lesioned at nucleus basalis magnocellularis (NBM), by treatment with choline pivaloyl ester (CPE). CPE was daily administered (60 micromol/Kg ip) during 3 weeks to rats selectively lesioned by AMPA infusion into right NBM; the intact left NBM serving as control. NGF levels were determined in cerebral cortex and hippocampus by Elisa assay. TrkA receptor expression was evaluated in right NBM by Western blotting analysis. CPE treatment significantly increased NGF levels in both hippocampus and neocortex in right NBM, compared with intact left counter-part and controls. Western blotting showed an evident enhancement in TrkA receptor expression in lesioned right NBM in comparison with intact left counter-part and controls. CPE treatment was also able to restore, in bilaterally NBM-lesioned rats, the disrupted cortical EEG and HVS activities as well as to reverse deficits in learning and memory in spatial navigation and probe trials, and cognitive capacities in object recognition task.
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Encéfalo/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Ciclopentanos/uso terapéutico , Trastornos de la Memoria/tratamiento farmacológico , Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Animales , Núcleo Basal de Meynert/efectos de los fármacos , Núcleo Basal de Meynert/patología , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Trastornos del Conocimiento/etiología , Electroencefalografía , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/etiología , Factor de Crecimiento Nervioso/genética , Pruebas Neuropsicológicas , Síndromes de Neurotoxicidad/complicaciones , Síndromes de Neurotoxicidad/patología , Ratas , Ratas Wistar , Tiempo de Reacción/efectos de los fármacos , Receptor trkA/genética , Factores de TiempoRESUMEN
BACKGROUND: In vitro and in vivo studies demonstrated the antiviral efficacy of nerve growth factor (NGF) and its cyto-protective effect in herpes simplex virus (HSV)-infected cells. The aims of this study were to evaluate the role of endogenous NGF in HSV corneal infection, and the effects of topical NGF treatment on herpetic keratitis. METHODS: Herpetic keratitis was induced in 40 rabbits with the HSV-1 McKrae strain. Animals were divided into four groups, and treated with topical neutralizing anti-NGF antibodies, NGF, acyclovir or balanced salt solution (BSS) respectively. The clinical course of HSV keratitis was evaluated and scored by slit-lamp examination. In addition, biochemical (immunohistochemistry for glycoprotein D) and molecular (nested PCR for glycoprotein D) analyses were carried out to estimate viral replication. RESULTS: Treatment with anti-NGF antibodies induced a more severe keratitis associated with increased biochemical and molecular markers of active viral replication. Two animals in this group developed lethal HSV encephalitis. Conversely, topical treatment with NGF induced a significant amelioration of clinical and laboratory parameters when compared to the BSS treated group (control). No significant differences were observed between NGF- and acyclovir-treated groups. CONCLUSIONS: This study demonstrated the crucial role of endogenous NGF in herpetic keratitis. The comparable effects of NGF and acyclovir confirm the antiviral activity of NGF, and indicate a potential use of topical NGF in herpetic keratitis.
Asunto(s)
Modelos Animales de Enfermedad , Queratitis Herpética/tratamiento farmacológico , Factor de Crecimiento Nervioso/uso terapéutico , Aciclovir/uso terapéutico , Administración Tópica , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antivirales/uso terapéutico , Citoprotección , Femenino , Herpesvirus Humano 1/fisiología , Técnicas para Inmunoenzimas , Queratitis Herpética/metabolismo , Factor de Crecimiento Nervioso/fisiología , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Conejos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Replicación ViralRESUMEN
BACKGROUND: HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC). Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC), on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS) in the imbalanced activity of glutamine synthase (GS), the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. RESULTS: Incubation of Lipari human cultured astroglial cells with gp 120 (0.1-10 nM) produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS). This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA). In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5-5 mM), dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. CONCLUSION: In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered glutamine release. All the effects of gp120 on astroglial cells were counteracted by NAC thus suggesting a novel and potentially useful approach in the treatment of glutammatergic disorders found in HAD patients.
Asunto(s)
Acetilcisteína/farmacología , Astrocitos/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Glutamato-Amoníaco Ligasa/fisiología , Proteína gp120 de Envoltorio del VIH/toxicidad , Análisis de Varianza , Astrocitoma/patología , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Interacciones Farmacológicas , Citometría de Flujo , Glutamina/metabolismo , Humanos , Etiquetado Corte-Fin in Situ/métodos , Masculino , Malondialdehído/metabolismo , Persona de Mediana EdadRESUMEN
The interleukin-2 is a cytokine that is essential for lymphocytic survival and function. Ectopic expression of the IL-2 receptor in epithelial tissues has been reported previously, although the functional significance of this expression is still being investigated. We provided novel structural and functional information on the expression of the IL-2 receptor in kidney cancer cells and in other normal and neoplastic human epithelial tissues. In A-498 kidney cancer cells, we showed that IL-2 binding to its own receptor triggers a signal transduction pathway leading to the inhibition of proliferation and apoptosis. We found that the inhibition of proliferation is associated with Erk1/2 dephosphorylation, whereas the survival signals appear to be mediated by Sgk1 activation. This investigation focuses on the IL-2 induced regulation of Sgk1 and describes a role of the IL-2 receptor and Sgk1 in the regulation of epithelial tumor cell death and survival.
Asunto(s)
Apoptosis , Proliferación Celular , Proteínas Inmediatas-Precoces/fisiología , Interleucina-2/fisiología , Neoplasias Renales/patología , Proteínas Serina-Treonina Quinasas/fisiología , Línea Celular Tumoral , Supervivencia Celular , Regulación de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Neoplasias Renales/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Interleucina-2/análisis , Transducción de SeñalRESUMEN
In our previous studies, we demonstrated that intraperitoneal (i.p.) injections with the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) stimulate the synthesis of the neurotrophin nerve growth factor (NGF) resulting in the structural and functional recovery of neuronal damage. This neurotrophin-mediated neuroprotective action of CCK-8 has opened a new perspective for a better understanding of the CCK neurobiological and pharmacological properties. To explore the possible beneficial effects of the CCK-induced increase of neurotrophin availability in brain, we compared the effects of i.p. CCK-8 in healthy rats and in a chemical kindling model using a subconvulsive dose of pentylenetetrazol (PTZ). Behavioural changes were monitored during treatment and classified according to a six-point scale. After 3 weeks of treatment (12 trials), the PTZ group of rats manifested generalized clonic-tonic seizures (Class 5 behaviour). For this reason, this time point was chosen to compare the effects of CCK-8 treatment on the expression of NGF, the brain derived neurotrophin factor (BDNF) and their receptors in the septum and hippocampus. We found that repeated i.p. injections with CCK-8 in adult rats result in: (1) an increase of NGF and BDNF protein and mRNA levels in the septum and hippocampus; (2) a down-regulation of TrkA and p75NTR and an up-regulation of TrkB; (3) reduced susceptibility to develop chemical kindling; (4) recovery of the PTZ-induced changes in the expression of neurotrophin receptors in the septal and hippocampal tissues. This data clearly indicates that CCK-induced variation of neurotrophin synthesis in brain is able to influence the susceptibility to develop seizures in adult rats most probably by counteracting the progressive neuronal dysfunction and/or damage.