RESUMEN
Mitotane (MTT) is an adrenolytic drug used in adjuvant and advanced treatments of adrenocortical carcinoma (ACC). Ionizing radiation (IR) is also used in adrenal cancer treatment, even though its biological action remains unknown. To provide a reliable in vivo preclinical model of ACC, we used mouse xenografts bearing human ACC to test the effects of MTT and IR alone and in combination. We evaluated tumor growth inhibition by the RECIST criteria and analyzed the cell cycle by flow cytometry (FCM). In the xenograft ACC model treated with MTT/IR in combination, we observed a marked inhibition of tumor growth, with strong tumor regression (p < 0.0001) compared to MTT and IR given alone (p < 0.05). The MTT results confirm its antisteroidogenic activity (p < 0.05) in the xenograft ACC model, revealing its ability to render cancer cells more prone to radiotherapy treatment. In addition, to explain the biological effect of these treatments on the Mismatch Repair System (MMR), we interfered with the MSH2 gene expression in untreated and MTT/IR-treated H295R and SW13 cell lines. Moreover, we observed that upon treatment with MTT/IR to induce DNA damage, MSH2 gene inhibition in both the H295R and SW13 cell lines did not allow DNA damage repair, thus inducing cell death. In conclusion, MTT seems to have a radiosensitizing property and, when given in combination with IR, is able to promote neoplastic growth inhibition, leading to a significant reduction in tumor size due to cell death.
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In an aging society, Alzheimer's disease (AD) exerts an increasingly serious health and economic burden. Current treatments provide inadequate symptomatic relief as several distinct pathological processes are thought to underlie the decline of cognitive and neural function seen in AD. This suggests that the efficacy of treatment requires a multitargeted approach. In this context, palmitoylethanolamide (PEA) provides a novel potential adjunct therapy that can be incorporated into a multitargeted treatment strategy. We used young (6-month-old) and adult (12-month-old) 3×Tg-AD mice that received ultramicronized PEA (um-PEA) for 3 months via a subcutaneous delivery system. Mice were tested with a range of cognitive and noncognitive tasks, scanned with magnetic resonance imaging/magnetic resonance spectroscopy (MRI/MRS), and neurochemical release was assessed by microdialysis. Potential neuropathological mechanisms were assessed postmortem by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and immunofluorescence. Our data demonstrate that um-PEA improves learning and memory, and ameliorates both the depressive and anhedonia-like phenotype of 3×Tg-AD mice. Moreover, it reduces Aß formation, the phosphorylation of tau proteins, and promotes neuronal survival in the CA1 subregion of the hippocampus. Finally, um-PEA normalizes astrocytic function, rebalances glutamatergic transmission, and restrains neuroinflammation. The efficacy of um-PEA is particularly potent in younger mice, suggesting its potential as an early treatment. These data demonstrate that um-PEA is a novel and effective promising treatment for AD with the potential to be integrated into a multitargeted treatment strategy in combination with other drugs. Um-PEA is already registered for human use. This, in combination with our data, suggests the potential to rapidly proceed to clinical use.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/deficiencia , Antiinflamatorios/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Etanolaminas/farmacología , Inflamación/tratamiento farmacológico , Aprendizaje/efectos de los fármacos , Trastornos de la Memoria/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Ácidos Palmíticos/farmacología , Proteínas tau/efectos de los fármacos , Factores de Edad , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Amidas , Animales , Antiinflamatorios/administración & dosificación , Conducta Animal/efectos de los fármacos , Región CA1 Hipocampal/inmunología , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiopatología , Disfunción Cognitiva/inmunología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Modelos Animales de Enfermedad , Etanolaminas/administración & dosificación , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/fisiopatología , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Trastornos de la Memoria/inmunología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Microdiálisis , Fármacos Neuroprotectores/administración & dosificación , Ácidos Palmíticos/administración & dosificaciónRESUMEN
BACKGROUND: The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. METHODS: Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. RESULTS: Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. CONCLUSIONS: Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.
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Hidrocarburos Aromáticos con Puentes/farmacología , Neuroglía/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Receptores CXCR4/genética , Tionas/farmacología , Fosfolipasas de Tipo C/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Metaboloma/genética , Neuroglía/efectos de los fármacos , Neuroglía/patología , Norbornanos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Tiocarbamatos , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Triple-negative breast cancer (TNBC), defined as lack of estrogen and progesterone receptors in the absence of protein overexpression/gene amplification of human epidermal growth factor receptor 2, is still a clinical challenge despite progress in breast cancer care. 1H magnetic resonance spectroscopy allows identification and non-invasive monitoring of TNBC metabolic aberrations and elucidation of some key mechanisms underlying tumor progression. Thus, it has the potential to improve in vivo diagnosis and follow-up and also to identify new targets for treatment. Several studies have shown an altered phosphatidylcholine (PtdCho) metabolism in TNBCs, both in patients and in experimental models. Upregulation of choline kinase-alpha, an enzyme of the Kennedy pathway that phosphorylates free choline (Cho) to phosphocholine (PCho), is a major contributor to the increased PCho content detected in TNBCs. Phospholipase-mediated PtdCho headgroup hydrolysis also contributes to the build-up of a PCho pool in TNBC cells. The oncogene-driven PtdCho cycle appears to be fine tuned in TNBC cells in at least three ways: by modulating the choline import, by regulating the activity or expression of specific metabolic enzymes, and by contributing to the rewiring of the entire metabolic network. Thus, only by thoroughly dissecting these mechanisms, it will be possible to effectively translate this basic knowledge into further development and implementation of Cho-based imaging techniques and novel classes of therapeutics.
RESUMEN
Tumor-specific immune tolerance represents an obstacle for the development of effective anti-tumor immune responses through cancer vaccines. We here evaluated the efficacy of chemo-immunotherapy in breaking tumor-specific immune tolerance in an almost incurable mouse model of spontaneous carcinogenesis.Transgenic HER-2/neu mice bearing large mammary tumors received the adoptive transfer of splenocytes and serum isolated from immune donors, with or without pre-conditioning with cyclophosphamide. Treatment efficacy was assessed by monitoring tumor growth by manual inspection and by magnetic resonance imaging. The same chemo-immunotherapy protocol was tested on tumor-free HER-2/neu mice, to evaluate the effects on tumor emergence.Our data show that chemo-immunotherapy hampered carcinogenesis and caused the regression of large mammary tumor lesions in tumor-bearing HER-2/neu mice. The complete eradication of a significant number of tumor lesions occurred only in mice receiving cyclophosphamide shortly before immunotherapy, and was associated with increased serum anti HER-2/p185 antibodies and tumor leukocyte infiltration. The same protocol significantly delayed the appearance of mammary tumors when administered to tumor-free HER-2/neu mice, indicating that this chemo-immunotherapy approach acted through the elicitation of an effective anti-tumor immune response. Overall, our data support the immune-modulatory role of chemotherapy in overcoming cancer immune tolerance when administered at lymphodepleting non-myeloablative doses shortly before transfer of antigen-specific immune cells and immunoglobulins. These findings open new perspectives on combining immune-modulatory chemotherapy and immunotherapy to overcome immune tolerance in cancer patients.
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Ciclofosfamida/administración & dosificación , Modelos Animales de Enfermedad , Inmunoterapia Adoptiva/métodos , Neoplasias Mamarias Experimentales/terapia , Animales , Antineoplásicos Alquilantes/administración & dosificación , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Ratones de la Cepa 129 , Ratones Transgénicos , Ratas , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. METHODS: The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. RESULTS: We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. CONCLUSIONS: Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.
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Neoplasias Encefálicas/tratamiento farmacológico , Quimiocina CXCL12/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Microglía/efectos de los fármacos , Péptidos/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Humanos , Ratones , Ratones Desnudos , Microglía/patología , Péptidos/farmacología , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastomas are the most common brain tumors in humans. Previously, we demonstrated that the muscarinic receptor agonist, arecaidine propargyl ester, via M2 receptors, inhibits cell proliferation in a time and dose-dependent manner and induces a severe apoptosis in human U251 and U87 glioblastoma cell lines. In order to clarify the mechanisms causing apoptosis after arecaidine treatment, we analyzed the ability of arecaidine to induce oxidative stress. By dichloro-dihydro-fluorescein diacetate (DCFDA) staining, we demonstrated that arecaidine increased the intracellular ROS levels. ROS accumulation was completely counteracted by the ROS scavenger, N-acetyl-l-cysteine (NAC). Apoptotic cell death appeared directly correlated to ROS production since NAC was able to counteract this effect. Although there was an up-regulation of some detoxifying enzyme expression such as superoxide dismutase (MnSOD) and sirtuin-1 (SIRT1), the cytotoxic effect caused by arecaidine treatment caused DNA damage, as demonstrated by the increase of histone γ-H2AX positive cells, and chromosomal aberrations. These effects were mediated by M2 receptor activation; in fact after silencing of M2 receptors by siRNA, the increase of γ-H2AX positive cells was abolished. In conclusion, in addition to a cytostatic effect previously described, in the present study we have better characterized the mechanisms causing the cytotoxic effects and the apoptotic cell death in glioblastoma cells after M2 receptor activation. These data allow to consider this receptor a new interesting therapeutic tool for the glioblastoma treatment.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Glioblastoma/metabolismo , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Acetilcisteína/farmacología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Humanos , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismoRESUMEN
Nanoparticle (NP)-based materials are promising agents for enhancing cancer diagnosis and treatment. Once functionalized for selective targeting of tumor-expressed molecules, they can specifically deliver drugs and diagnostic molecules inside tumor cells. In the present work, we evaluated the in vivo melanoma-targeting ability of a nanovector (HFt-MSH-PEG) based on human protein ferritin (HFt), functionalized with both melanoma-targeting melanoma stimulating hormone (α-MSH) and stabilizing poly(ethylene glycol) (PEG) molecules. Independent and complementary techniques, such as whole-specimen confocal microscopy and magnetic resonance imaging, were used to detect in vivo localization of NP constructs with suitable tracers (i.e., fluorophores or magnetic metals). Targeted HFt-MSH-PEG NPs accumulated persistently at the level of primary melanoma and with high selectivity with respect to other organs. Melanoma localization of untargeted HFt-PEG NPs, which lack the α-MSH moiety, was less pronounced. Furthermore, HFt-MSH-PEG NPs accumulated to a significantly lower extent and with a different distribution in a diverse type of tumor (TS/A adenocarcinoma), which does not express α-MSH receptors. Finally, in a spontaneous lung metastasis model, HFt-MSH-PEG NPs localized at the metastasis level as well. These results suggest that HFt-MSH-PEG NPs are suitable carriers for selective in vivo delivery of diagnostic or therapeutic agents to cutaneous melanoma.
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Imagen por Resonancia Magnética/métodos , Melanoma/patología , Nanocápsulas/química , Neoplasias Cutáneas/patología , alfa-MSH/farmacocinética , Animales , Línea Celular Tumoral , Medios de Contraste/síntesis química , Colorantes Fluorescentes/síntesis química , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , Nanocápsulas/ultraestructura , Tamaño de la Partícula , Neoplasias Cutáneas/metabolismoRESUMEN
We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC.
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Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Ciclopentanos/farmacología , Quinolinas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Adolescente , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/genética , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Adulto , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Daño del ADN , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Diesel exhaust particles (DEP) are major constituents of ambient air pollution and their adverse health effect is an area of intensive investigations. With respect to the immune system, DEP have attracted significant research attention as a factor that could influence allergic diseases interfering with cytokine production and chemokine expression. With this exception, scant data are available on the impact of DEP on lymphocyte homeostasis. Here, the effects of nanoparticles from Euro 4 (E4) and Euro 5 (E5) light duty diesel engines on the phenotype and function of T lymphocytes from healthy donors were evaluated. METHODS: T lymphocytes were isolated from peripheral blood obtained from healthy volunteers and subsequently stimulated with different concentration (from 0.15 to 60 µg/ml) and at different time points (from 24 h to 9 days) of either E4 or E5 particles. Immunological parameters, including apoptosis, autophagy, proliferation levels, mitochondrial function, expression of activation markers and cytokine production were evaluated by cellular and molecular analyses. RESULTS: DEP exposure caused a pronounced autophagic-lysosomal blockade, thus interfering with a key mechanism involved in the maintaining of T cell homeostasis. Moreover, DEP decreased mitochondrial membrane potential but, unexpectedly, this effect did not result in changes of the apoptosis and/or necrosis levels, as well as of intracellular content of adenosine triphosphate (ATP). Finally, a down-regulation of the expression of the alpha chain of the interleukin (IL)-2 receptor (i.e., the CD25 molecule) as well as an abnormal Th1 cytokine expression profile (i.e., a decrease of IL-2 and interferon (IFN)-γ production) were observed after DEP exposure. No differences between the two compounds were detected in all studied parameters. CONCLUSIONS: Overall, our data identify functional and phenotypic T lymphocyte parameters as relevant targets for DEP cytotoxicity, whose impairment could be detrimental, at least in the long run, for human health, favouring the development or the progression of diseases such as autoimmunity and cancer.
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Contaminantes Atmosféricos/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Hollín/toxicidad , Linfocitos T/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Adulto , Contaminantes Atmosféricos/química , Contaminantes Atmosféricos/metabolismo , Autofagia/efectos de los fármacos , Transporte Biológico , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Cinética , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Tamaño de la Partícula , Hollín/química , Hollín/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/ultraestructura , Emisiones de Vehículos/análisis , Adulto JovenRESUMEN
The mineralocorticoid receptor (MR) controls adipocyte function, but its role in the conversion of white adipose tissue (WAT) into thermogenic fat has not been elucidated. We investigated responses to the MR antagonists spironolactone (spiro; 20 mg/kg/d) and drospirenone (DRSP; 6 mg/kg/d) in C57BL/6 mice fed a high-fat (HF) diet for 90 d. DRSP and spiro curbed HF diet-induced impairment in glucose tolerance, and prevented body weight gain and white fat expansion. Notably, either MR antagonist induced up-regulation of brown adipocyte-specific transcripts and markedly increased protein levels of uncoupling protein 1 (UCP1) in visceral and inguinal fat depots when compared with the HF diet group. Positron emission tomography and magnetic resonance spectroscopy confirmed acquisition of brown fat features in WAT. Interestingly, MR antagonists markedly reduced the autophagic rate both in murine preadipocytes in vitro (10(-5) M) and in WAT depots in vivo, with a concomitant increase in UCP1 protein expression. Moreover, the autophagy repressor bafilomycin A1 (10(-8) M) mimicked the effect of MR antagonists, increasing UCP1 protein expression in primary preadipocytes. Hence, we showed that adipocyte MR regulates brown remodeling of WAT through a modulation of autophagy. These results provide a rationale for the use of MR antagonists to prevent the adverse metabolic consequences of adipocyte dysfunction.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Androstenos/farmacología , Autofagia/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Intolerancia a la Glucosa/prevención & control , Antagonistas de Receptores de Mineralocorticoides/farmacología , Receptores de Mineralocorticoides/fisiología , Espironolactona/farmacología , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Adipogénesis/fisiología , Aldosterona/farmacología , Androstenos/uso terapéutico , Animales , Composición Corporal/efectos de los fármacos , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Intolerancia a la Glucosa/etiología , Conducto Inguinal , Grasa Intraabdominal/efectos de los fármacos , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Macrólidos/farmacología , Ratones , Ratones Endogámicos C57BL , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Obesidad/tratamiento farmacológico , Obesidad/fisiopatología , Obesidad/prevención & control , Receptores de Mineralocorticoides/efectos de los fármacos , Espironolactona/uso terapéutico , Proteína Desacopladora 1 , Regulación hacia Arriba/efectos de los fármacos , Aumento de Peso/efectos de los fármacosRESUMEN
CONTEXT: Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis and no effective treatment. ACC is characterized by an increased production of IGF-II and by estrogen receptor (ER)-α up-regulation. OBJECTIVE: The objective of this study was to define the role played by ERα in 17ß-estradiol (E2)- and IGF-II-dependent ACC growth and evaluate whether selective estrogen receptor modulators are effective in controlling ACC growth in vivo. EXPERIMENTAL DESIGN: The human adrenocortical cell line H295R was used as an in vitro model and to generate xenograft tumors in athymic nude mice. RESULTS: In H295R cells IGF-II controlled expression of steroidogenic factor-1 that, in turn, increased aromatase transcription and, consequently, estrogen production, inducing cell proliferation. ERα silencing significantly blocked E2- and IGF-II-dependent cell proliferation. This effect was dependent on the regulation of cyclin D1 expression by ERα, activated in response to both E2 and IGF-II. In fact, IGF-II induced ERα activation by phosphorylating serine 118 and 167. Furthermore, we demonstrated that ERα mediated E2-induced nongenomic signaling that stimulated IGF-I receptor (IGF1R), ERK1/2, and AKT phosphorylation, resulting in a ligand-independent activation of the IGF1R-induced pathway. In addition, E2 potentiated this pathway by up-regulating IGF1R expression as a consequence of increased cAMP-responsive element binding protein activation and binding to IGF1R promoter. The estrogen antagonist, hydroxytamoxifen, the active metabolite of tamoxifen, reduced IGF1R protein levels and both E2- and IGF-II-induced cell proliferation. Moreover, H295R xenograft growth was strongly reduced by tamoxifen. CONCLUSION: These findings establish a critical role for ERα in E2- and IGF-II-dependent ACC proliferation and provide a rationale for targeting ERα to control the proliferation of ACC.
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Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Antineoplásicos Hormonales/farmacología , Carcinoma/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Animales , Antineoplásicos Hormonales/uso terapéutico , Carcinoma/genética , Carcinoma/patología , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody - directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. RESULTS: An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. CONCLUSION: The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.
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Anticuerpos Monoclonales/inmunología , Citosina Desaminasa/inmunología , Proteínas Fúngicas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/genética , Citosina Desaminasa/genética , Proteínas Fúngicas/genética , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Gemcitabine (dFdCyd) is a deoxycytidine analogue showing a broad spectrum of cytotoxic activity; additionally, at non-cytotoxic concentrations, it is a potent radiosensitiser. Its in vitro and in vivo effects were studied on C6 rat glioma. In vitro, dFdCyd induced an increase in S-phase cells at the end of treatment, with a reduction in G1 and G2 cell cycle-phase cells and relevant effects on the apoptotic rate. The in vivo effects of dFdCyd were studied on rats bearing intracranial C6 glioma. The drug was administered at a dose of 120 mg/Kg every 3 days x 4 doses. A significant effect on tumour growth was detected by longitudinal MRI analyses. Furthermore, the drug induced an inhibitory effect on tumour growth, 72 h after a 300 mg/Kg single dose. Analyses performed on tumour specimens showed relevant G1-phase accumulation and about 45% apoptotic cells. The present results justify further studies to determine the potential efficacy of dFdCyd in the treatment of malignant gliomas.
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Antimetabolitos Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Glioma/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de los fármacos , Desoxicitidina/farmacología , Glioma/patología , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Wistar , GemcitabinaRESUMEN
U87MG human glioma cells in cultures expressed metabotropic glutamate (mGlu) receptors mGlu2 and mGlu3. Addition of the mGlu2/3 receptor antagonist LY341495 to the cultures reduced cell growth, expression of cyclin D1/2, and activation of the MAP kinase and phosphatidylinositol-3-kinase pathways. This is in line with the evidence that activation of mGlu2/3 receptors sustains glioma cell proliferation. U87MG cells were either implanted under the skin (1x10(6) cells/0.5 ml) or infused into the caudate nucleus (0.5x10(6) cells/5 microl) of nude mice. Animals were treated for 28 days with mGlu receptor antagonists by means of subcutaneous osmotic minipumps. Treatments with LY341495 or (2S)-alpha-ethylglutamate (both infused at a rate of 1 mg/kg per day) reduced the size of tumors growing under the skin. Infusion of LY341495 (10 mg/kg per day) also reduced the growth of brain tumors, as assessed by magnetic resonance imaging analysis carried out every seven days. The effect of drug treatment was particularly evident during the exponential phase of tumor growth, that is, between the third and the fourth week following cell implantation. Immunohistochemical analysis showed that U87MG cells retained the expression of mGlu2/3 receptors when implanted into the brain of nude mice. These data suggest that mGlu2/3 receptor antagonists are of potential use in the experimental treatment of malignant gliomas.
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Aminoácidos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Antagonistas de Aminoácidos Excitadores/farmacología , Glioma/tratamiento farmacológico , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Xantenos/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/biosíntesis , Ciclina D1/efectos de los fármacos , Ciclina D2 , Ciclinas/biosíntesis , Ciclinas/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Inmunohistoquímica , Antígeno Ki-67/efectos de los fármacos , Antígeno Ki-67/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Imagen por Resonancia Magnética , Ratones , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Controversy exists whether the development of left-ventricular hypertrophy (LVH) is a mechanism able to prevent cardiac dysfunction under conditions of pressure overload. In the present study we re-assessed the long-term effects of attenuating LVH by using L- and D-propranolol, which are equally able to inhibit the development of LVH induced by aortic banding. The aortic arch was banded proximal to the left common carotid artery in 71 CD-1 mice that were then assigned randomly to receive L-propranolol, D-propranolol (both 80 mg/kg per day) or vehicle. Concurrently, sham-operated mice were given L-propranolol, D-propranolol or vehicle. LV dimension and performance were evaluated under isoflurane anaesthesia by cine-magnetic resonance imaging, echocardiography and cardiac catheterization up to 8 weeks after surgery. After 2 weeks of pressure overload, the vehicle-treated banded mice had enhanced LV weight, normal chamber size and increased relative wall thickness (concentric hypertrophy), whereas L-propranolol- or D-propranolol-banded mice showed a markedly blunted hypertrophic response, i.e. normal chamber size and normal relative wall thickness, as well as preserved systolic LV chamber function. After 4 weeks, the vehicle-treated banded mice showed LV enlargement with a reduced relative wall thickness (eccentric remodelling) and a clear-cut deterioration in LV systolic function. In contrast, L-propranolol- or D-propranolol-treated banded mice showed normal chamber size with a normal relative wall thickness and preserved systolic function. A distinct histological feature was that in banded mice, L-or D-propranolol attenuated the development of cardiomyocyte hypertrophy but not the attendant myocardial fibrosis. At the 8-week stage, LV dysfunction was present in propranolol-treated banded mice although it was much less severe than in vehicle-treated banded mice. It is concluded that (i) deterioration of LV systolic performance is delayed if LV hypertrophy is inhibited, (ii) banding-induced deterioration of LV systolic function is associated with LV eccentric remodelling and (iii) the antihypertrophic effect of propranolol is due to a selective action on cardiomyocytes rather than on collagen accumulation