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Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+ T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates.
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Amilorida/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/uso terapêutico , Esomeprazol/uso terapêutico , Inibidores da Bomba de Prótons/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Amilorida/uso terapêutico , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos BALB C , Trocador 1 de Sódio-Hidrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Macrófagos Associados a Tumor/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Recent studies reported that the uptake of [18F]-fluorodeoxyglucose (FDG) is increased in the spinal cord (SC) and decreased in the motor cortex (MC) of patients with ALS, suggesting that the disease might differently affect the two nervous districts with different time sequence or with different mechanisms. Here we show that MC and SC astrocytes harvested from newborn B6SJL-Tg (SOD1G93A) 1Gur mice could play different roles in the pathogenesis of the disease. Spectrophotometric and cytofluorimetric analyses showed an increase in redox stress, a decrease in antioxidant capacity and a relative mitochondria respiratory uncoupling in MC SOD1G93A astrocytes. By contrast, SC mutated cells showed a higher endurance against oxidative damage, through the increase in antioxidant defense, and a preserved respiratory function. FDG uptake reproduced the metabolic response observed in ALS patients: SOD1G93A mutation caused a selective enhancement in tracer retention only in mutated SC astrocytes, matching the activity of the reticular pentose phosphate pathway and, thus, of hexose-6P dehydrogenase. Finally, both MC and SC mutated astrocytes were characterized by an impressive ultrastructural enlargement of the endoplasmic reticulum (ER) and impairment in ER-mitochondria networking, more evident in mutated MC than in SC cells. Thus, SOD1G93A mutation differently impaired MC and SC astrocyte biology in a very early stage of life.
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Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs. The oxidation of HMGB1 cysteines switches its extracellular activities from the orchestration of tissue regeneration to the exacerbation of inflammation. Extracellular HMGB1 is present at high amount and undergoes oxidation in patients with MDs and in mouse models of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy 3 (LGMDR3) compared to controls. Genetic ablation of HMGB1 in muscles of DMD mice leads to an amelioration of the dystrophic phenotype as evidenced by the reduced inflammation and muscle degeneration, indicating that HMGB1 oxidation is a detrimental process in MDs. Pharmacological treatment with an engineered nonoxidizable variant of HMGB1, called 3S, improves functional performance, muscle regeneration, and satellite cell engraftment in dystrophic mice while reducing inflammation and fibrosis. Overall, our data demonstrate that the balance between HMGB1 redox isoforms dictates whether skeletal muscle is in an inflamed or regenerating state, and that the nonoxidizable form of HMGB1 is a possible therapeutic approach to counteract the progression of the dystrophic phenotype. Rebalancing the HMGB1 redox isoforms may also be a therapeutic strategy for other disorders characterized by chronic oxidative stress and inflammation.
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Proteína HMGB1 , Distrofia Muscular de Duchenne , Animais , Proteína HMGB1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Oxirredução , Isoformas de Proteínas/metabolismoRESUMO
Boosting antitumor immunity has emerged as a powerful strategy in cancer treatment. While releasing T-cell brakes has received most attention, tumor recognition by T cells is a pre-requisite. Radiotherapy and certain cytotoxic drugs induce the release of damage-associated molecular patterns, which promote tumor antigen cross-presentation and T-cell priming. Antibodies against the "do not eat me" signal CD47 cause macrophage phagocytosis of live tumor cells and drive the emergence of antitumor T cells. Here we show that CXCR4 activation, so far associated only with tumor progression and metastasis, also flags tumor cells to immune recognition. Both CXCL12, the natural CXCR4 ligand, and BoxA, a fragment of HMGB1, promote the release of DAMPs and the internalization of CD47, leading to protective antitumor immunity. We designate as Immunogenic Surrender the process by which CXCR4 turns in tumor cells to macrophages, thereby subjecting a rapidly growing tissue to immunological scrutiny. Importantly, while CXCL12 promotes tumor cell proliferation, BoxA reduces it, and might be exploited for the treatment of malignant mesothelioma and a variety of other tumors.
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Antígeno CD47 , Mesotelioma , Animais , Linhagem Celular Tumoral , Imunização , Macrófagos , Mesotelioma/imunologia , Mesotelioma/metabolismo , Mesotelioma/terapia , Camundongos , FagocitoseRESUMO
In cognitively normal patients, mild hyperglycemia selectively decreases 18F-Fluorodeoxyglucose (FDG) uptake in the posterior brain, reproducing Alzheimer disease pattern, hampering the diagnostic accuracy of this widely used tool. This phenomenon might involve either a heterogeneous response of glucose metabolism or a different sensitivity to hyperglycemia-related redox stress. Indeed, previous studies reported a close link between FDG uptake and activation of a specific pentose phosphate pathway (PPP), triggered by hexose-6P-dehydrogenase (H6PD) and contributing to fuel NADPH-dependent antioxidant responses in the endoplasmic reticulum (ER). To clarify this issue, dynamic positron emission tomography was performed in 40 BALB/c mice four weeks after administration of saline (n = 17) or 150 mg/kg streptozotocin (n = 23, STZ). Imaging data were compared with biochemical and histological indexes of glucose metabolism and redox balance. Cortical FDG uptake was homogeneous in controls, while it was selectively decreased in the posterior brain of STZ mice. This difference was independent of the activity of enzymes regulating glycolysis and cytosolic PPP, while it was paralleled by a decreased H6PD catalytic function and enhanced indexes of oxidative damage. Thus, the relative decrease in FDG uptake of the posterior brain reflects a lower activation of ER-PPP in response to hyperglycemia-related redox stress in these areas.
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Encéfalo/patologia , Diabetes Mellitus Experimental/fisiopatologia , Retículo Endoplasmático/patologia , Fluordesoxiglucose F18/metabolismo , Glicólise , Hiperglicemia/complicações , Tomografia por Emissão de Pósitrons/métodos , Animais , Transporte Biológico , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Retículo Endoplasmático/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Via de Pentose Fosfato , Compostos Radiofarmacêuticos/metabolismoRESUMO
BACKGROUND: We recently reported that enhanced [18F]-fluorodeoxyglucose (FDG) uptake in skeletal muscles predicts disease aggressiveness in patients with amyotrophic lateral sclerosis (ALS). The present experimental study aimed to assess whether this predictive potential reflects the link between FDG uptake and redox stress that has been previously reported in different tissues and disease models. METHODS: The study included 15 SOD1G93A mice (as experimental ALS model) and 15 wildtype mice (around 120 days old). Mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested quadriceps and hearts by biochemical, immunohistochemical, and immunofluorescence analysis. Colocalization between the endoplasmic reticulum (ER) and the fluorescent FDG analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was performed in fresh skeletal muscle sections. Finally, mitochondrial ultrastructure and bioenergetics were evaluated in harvested quadriceps and hearts. RESULTS: FDG retention was significantly higher in hindlimb skeletal muscles of symptomatic SOD1G93A mice with respect to control ones. This difference was not explained by any acceleration in glucose degradation through glycolysis or cytosolic pentose phosphate pathway (PPP). Similarly, it was independent of inflammatory infiltration. Rather, the high FDG retention in SOD1G93A skeletal muscle was associated with an accelerated generation of reactive oxygen species. This redox stress selectively involved the ER and the local PPP triggered by hexose-6P-dehydrogenase. ER involvement was confirmed by the colocalization of the 2-NBDG with a vital ER tracker. The oxidative damage in transgenic skeletal muscle was associated with a severe impairment in the crosstalk between ER and mitochondria combined with alterations in mitochondrial ultrastructure and fusion/fission balance. The expected respiratory damage was confirmed by a deceleration in ATP synthesis and oxygen consumption rate. These same abnormalities were represented to a markedly lower degree in the myocardium, as a sample of non-voluntary striated muscle. CONCLUSION: Skeletal muscle of SOD1G93A mice reproduces the increased FDG uptake observed in ALS patients. This finding reflects the selective activation of the ER-PPP in response to significant redox stress associated with alterations of mitochondrial ultrastructure, networking, and connection with the ER itself. This scenario is less severe in cardiomyocytes suggesting a relevant role for either communication with synaptic plaque or contraction dynamics.
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This study aims to verify in experimental models of hyperglycemia induced by streptozotocin (STZ-DM) to what degree the high competition between unlabeled glucose and metformin (MET) treatment might affect the accuracy of cancer FDG imaging. The study included 36 "control" and 36 "STZ-DM" Balb/c mice, undergoing intraperitoneal injection of saline or streptozotocin, respectively. Two-weeks later, mice were subcutaneously implanted with breast (4 T1) or colon (CT26) cancer cells and subdivided in three subgroups for treatment with water or with MET at 10 or 750 mg/Kg/day. Two weeks after, mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested tumors. Finally, competition by glucose, 2-deoxyglucose (2DG) and the fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) on FDG uptake was studied in 4 T1 and CT26 cultured cells. STZ-DM slightly decreased cancer volume and FDG uptake rate (MRF). More importantly, it also abolished MET capability to decelerate lesion growth and MRF. This metabolic reprogramming closely agreed with the activity of hexose-6-phosphate dehydrogenase within the endoplasmic reticulum. Finally, co-incubation with 2DG virtually abolished FDG and 2-NBDG uptake within the endoplasmic reticulum in cultured cells. These data challenge the current dogma linking FDG uptake to glycolytic flux and introduce a new model to explain the relation between glucose analogue uptake and hexoses reticular metabolism. This selective fate of FDG contributes to the preserved sensitivity of PET imaging in oncology even in chronic moderate hyperglycemic conditions.
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OBJECTIVES: The present study aims to verify the relationship between glucose consumption and uptake of 18F-2-deoxy-glucose (FDG) in the skeletal muscle (SM) of experimental models of streptozotocin-induced diabetes mellitus (STZ-DM). METHODS: The study included 36 Balb/c mice. Two weeks after intraperitoneal administration of saline (control group, n = 18) or 150 mg streptozotocin (STZ-DM group, n = 18), the two cohorts were submitted to an oral glucose tolerance test and were further subdivided into three groups (n = 6 each): untreated and treated with metformin (MTF) at low or high doses (10 or 750 mg/kg daily, respectively). Two weeks thereafter, all mice were submitted to dynamic micro-positron emission tomography (PET) imaging after prolonged fasting. After sacrifice, enzymatic pathways and response to oxidative stress were evaluated in harvested SM. RESULTS: On PET imaging, the FDG uptake rate in hindlimb SM was significantly lower in nondiabetic mice as compared with STZ-DM-untreated mice. MTF had no significant effect on SM FDG uptake in untreated mice; however, its high dose induced a significant decrease in STZ-DM animals. Upon conventional analysis, the SM standard uptake value was higher in STZ-DM mice, while MTF was virtually ineffective in either control or STZ-DM models. This metabolic reprogramming was not explained by any change in cytosolic glucose metabolism. By contrast, it closely agreed with the catalytic function of hexose-6P-dehydrogenase (H6PD; i.e., the trigger of a specific pentose phosphate pathway selectively located within the endoplasmic reticulum). In agreement with this role, the H6PD enzymatic response to both STZ-DM and MTF matched the activation of the NADPH-dependent antioxidant responses to the increased generation of reactive oxygen species caused by chronic hyperglycemia. Ex vivo analysis of tracer kinetics confirmed that the enhanced SM avidity for FDG occurred despite a significant reduction in glucose consumption, while it was associated with increased radioactivity transfer to the endoplasmic reticulum. CONCLUSIONS: These data challenge the current dogma linking FDG uptake to the glycolytic rate. They instead introduce a new model considering a strict link between the uptake of this glucose analog, H6PD reticular activity, and oxidative damage in diabetes, at least under fasting condition.
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Diabetes Mellitus Experimental/metabolismo , Fluordesoxiglucose F18/metabolismo , Músculo Esquelético/metabolismo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/diagnóstico por imagem , Jejum , Teste de Tolerância a Glucose , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/diagnóstico por imagem , Estresse Oxidativo , Tomografia por Emissão de Pósitrons , Estreptozocina/administração & dosagemRESUMO
BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NOD-like receptor family, pyrin domain containing 3 (NLRP3) gene, which cause uncontrolled IL-1ß secretion. Proton pump inhibitors (PPIs), which are commonly used as inhibitors of gastric acid production, also have anti-inflammatory properties, protect mice from sepsis, and prevent IL-1ß secretion by monocytes from patients with CAPS. OBJECTIVE: We sought to develop a novel Nlrp3 knock-in (KI) mouse model of CAPS to study amyloidosis, a severe CAPS complication, and test novel therapeutic approaches. METHODS: We generated KI mice by engineering the N475K mutation, which is associated with the CAPS phenotype, into the mouse Nlrp3 gene. KI and wild-type mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. RESULTS: Mutant Nlrp3 KI mice displayed features that recapitulate the immunologic and clinical phenotype of CAPS. They showed systemic inflammation with high levels of serum proinflammatory cytokines, inflammatory infiltrates in various organs, and amyloid deposits in the spleen, liver, and kidneys. Toll-like receptor stimulated macrophages from KI mice secreted high levels of IL-1ß, IL-18, and IL-1α but low amounts of IL-1 receptor antagonist. Treatment of KI mice with PPIs had a clear clinical effect, showing a reduction in inflammatory manifestations, regression of amyloid deposits, and normalization of proinflammatory and anti-inflammatory cytokine production by macrophages. CONCLUSION: Nlrp3 KI mice displayed a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness of PPIs associated with a lack of toxicity indicates that these drugs could represent relevant adjuvants to the anti-IL-1 drugs in patients with CAPS and other IL-1-driven diseases.
Assuntos
Amiloidose , Síndromes Periódicas Associadas à Criopirina , Proteína 3 que Contém Domínio de Pirina da Família NLR , Inibidores da Bomba de Prótons/farmacologia , Amiloidose/tratamento farmacológico , Amiloidose/genética , Amiloidose/imunologia , Animais , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Síndromes Periódicas Associadas à Criopirina/genética , Síndromes Periódicas Associadas à Criopirina/imunologia , Síndromes Periódicas Associadas à Criopirina/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Camundongos , Camundongos Mutantes , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
BACKGROUND: Oxidative stress and its interference on myocardial metabolism play a major role in Doxorubicin (DXR) cardiotoxic cascade. METHODS: Mice models of neuroblastoma (NB) were treated with 5 mg DXR/kg, either free (Free-DXR) or encapsulated in untargeted (SL[DXR]) or in NB-targeting Stealth Liposomes (pep-SL[DXR] and TP-pep-SL[DXR]). Control mice received saline. FDG-PET was performed at baseline (PET1) and 7 days after therapy (PET2). At PET2 Troponin-I and NT-proBNP were assessed. Explanted hearts underwent biochemical, histological, and immunohistochemical analyses. Finally, FDG uptake and glucose consumption were simultaneously measured in cultured H9c2 in the presence/absence of Free-DXR (1 µM). RESULTS: Free-DXR significantly enhanced the myocardial oxidative stress. Myocardial-SUV remained relatively stable in controls and mice treated with liposomal formulations, while it significantly increased at PET2 with respect to baseline in Free-DXR. At this timepoint, myocardial-SUV was directly correlated with both myocardial redox stress and hexose-6-phosphate-dehydrogenase (H6PD) enzymatic activity, which selectively sustain cellular anti-oxidant mechanisms. Intriguingly, in vitro, Free-DXR selectively increased FDG extraction fraction without altering the corresponding value for glucose. CONCLUSION: The direct correlation between cardiac FDG uptake and oxidative stress indexes supports the potential role of FDG-PET as an early biomarker of DXR oxidative damage.
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Doxorrubicina/química , Fluordesoxiglucose F18/farmacocinética , Coração/efeitos dos fármacos , Miocárdio/patologia , Estresse Oxidativo , Animais , Antioxidantes , Biomarcadores/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Glucose/química , Glucose/farmacocinética , Humanos , Imuno-Histoquímica , Cinética , Camundongos , Camundongos Nus , Neuroblastoma/tratamento farmacológico , Oxirredução , Tomografia por Emissão de PósitronsRESUMO
PURPOSE: The endoplasmic reticulum (ER) contains hexose-6P-dehydrogenase (H6PD). This enzyme competes with glucose-6P-phosphatase for processing a variety of phosphorylated hexoses including 2DG-6P. The present study aimed to verify whether this ER glucose-processing machinery contributes to brain FDG uptake. METHODS: Effect of the H6PD inhibitor metformin on brain 18F-FDG accumulation was studied, in vivo, by microPET imaging. These data were complemented with the in vitro estimation of the lumped constant (LC). Finally, reticular accumulation of the fluorescent 2DG analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2NBDG) and its response to metformin was studied by confocal microscopy in cultured neurons and astrocytes. RESULTS: Metformin halved brain 18F-FDG accumulation without altering whole body tracer clearance. Ex vivo, this same response faced the doubling of both glucose consumption and lactate release. The consequent fall in LC was not explained by any change in expression or activity of its theoretical determinants (GLUTs, hexokinases, glucose-6P-phosphatase), while it agreed with the drug-induced inhibition of H6PD function. In vitro, 2NBDG accumulation selectively involved the ER lumen and correlated with H6PD activity being higher in neurons than in astrocytes, despite a lower glucose consumption. CONCLUSIONS: The activity of the reticular enzyme H6PD profoundly contributes to brain 18F-FDG uptake. These data challenge the current dogma linking 2DG/FDG uptake to the glycolytic rate and introduce a new model to explain the link between 18-FDG uptake and neuronal activity.
Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Retículo Endoplasmático/metabolismo , Fluordesoxiglucose F18/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Desidrogenases de Carboidrato/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxirredução/efeitos dos fármacos , Tomografia por Emissão de PósitronsRESUMO
Neoplastic cells live in a stressful context and survive thanks to their ability to overcome stress. Thus, tumor cell responses to stress are potential therapeutic targets. We selected two such responses in melanoma and sarcoma cells: the xc- antioxidant system, that opposes oxidative stress, and surface v-ATPases that counteract the low pHi by extruding protons, and targeted them with the xc- blocker sulfasalazine and the proton pump inhibitor esomeprazole. Sulfasalazine inhibited the cystine/cysteine redox cycle and esomeprazole decreased pHi while increasing pHe in tumor cell lines. Although the single treatment with either drug slightly inhibited cell proliferation and motility, the association of sulfasalazine and esomeprazole powerfully decreased sarcoma and melanoma growth and migration. In the 3-methylcholanthrene (3-MCA)-induced sarcoma model, the combined therapy strongly reduced the tumor burden and increased the survival time: notably, 22 % of double-treated mice recovered and survived off therapy. Tumor-associated macrophages (TAM) displaying M2 markers, that abundantly infiltrate sarcoma and melanoma, overexpress xc- and membrane v-ATPases and were drastically decreased in tumors from mice undergone the combined therapy. Thus, the double targeting of tumor cells and macrophages by sulfasalazine and esomeprazole has a double therapeutic effect, as decreasing TAM infiltration deprives tumor cells of a crucial allied. Sulfasalazine and esomeprazole may therefore display unexpected therapeutic values, especially in case of hard-to-treat cancers.
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IL-1ß acts in concert with anti-inflammatory cytokines, in particular, IL-1R antagonist (IL-1Ra), to ensure the correct development and outcome of the inflammation: imbalance in the IL-1ß/IL-1Ra ratio is implicated in many human diseases and may lead to dramatic consequences. In this article, we show that single TLR engagement induces IL-1ß and, with a little delay, IL-1Ra. Differently, costimulation of TLR2, TLR4, and TLR7/8 enhances IL-1ß secretion but severely inhibits IL-1Ra production. The IL-1ß/IL-1Ra unbalance after activation of multiple TLRs depends on the insurgence of oxidative stress, because of enhanced production of reactive oxygen species and failure of the antioxidant systems. Increased reactive oxygen species levels increase ATP externalization by monocytes, resulting in enhanced inflammasome activation and IL-1ß secretion. Oxidative stress then induces cell responses to stress, including inhibition of protein synthesis, which, in turn, is responsible for the impaired production of IL-1Ra. IL-1Ra secretion is restored by exogenous antioxidants that oppose oxidative stress. Similar effects are evident also on other cytokines: TNF-α is induced, whereas IL-6 is inhibited by costimulation. Our findings provide a molecular basis to the imbalance between proinflammatory and regulatory cytokine circuits that occur in various pathologic conditions, and suggest new strategies for controlling inflammation.
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Anti-Inflamatórios/imunologia , Mediadores da Inflamação/imunologia , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Estresse Oxidativo/imunologia , Receptores Toll-Like/imunologia , Citocinas/imunologia , Feminino , Humanos , Inflamassomos/imunologia , Masculino , Espécies Reativas de Oxigênio/imunologia , Receptores Toll-Like/agonistasRESUMO
SIGNIFICANCE: Increasing evidence indicates that cancer development and progression are promoted by the joint action of redox distress and inflammation, supporting the potential impact of therapies aimed at restoring the redox homeostasis and fighting inflammation. RECENT ADVANCES: Most of the literature of the last 40 years converges to the view that continuous oxidative stress and chronic inflammation sustain each other, leads to transformation of a normal cell to a neoplastic cell, and promotes tumor progression. Some recent findings, however, support an alternative model whereby the increased production of reactive oxygen species (ROS) is an attempt to defend more than a pathogenetic factor in cancer. Rather, tumor development and progression may be promoted by an excess of antioxidants, induced in both transformed cells and recruited inflammatory cells as an adaptive response to ROS. CRITICAL ISSUES: Although the link among redox stress, chronic inflammation, and cancer is widely recognized, the underlying mechanisms are far to be understood. The redox unbalance of the microenvironment is likely to modulate the bioactivity of damage-associated molecular pattern molecules such as HMGB1, which are released by stressed tissues and play pleiotropic functions on tumor and inflammatory cells, but how this occur, and the relevant consequences, are still unclear. FUTURE DIRECTIONS: In vivo measurement of cell redox status is an important challenge for future investigations. The improvement of the methodologies for ROS and antioxidant detection will allow a better understanding of the redox-related events in the tumor microenvironment with tremendous application potential in the development of rational combination therapies for cancer treatment.
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Carcinogênese/patologia , Inflamação/patologia , Neoplasias/patologia , Transdução de Sinais/fisiologia , Animais , Antioxidantes/metabolismo , Progressão da Doença , Humanos , Oxirredução , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
L19-tumor necrosis factor alpha (L19mTNF-α; L), a fusion protein consisting of mouse TNFα and the human antibody fragment L19 directed to the extra domain-B (ED-B) of fibronectin, is able to selectively target tumor vasculature and to exert a long-lasting therapeutic activity in combination with melphalan (M) in syngeneic mouse tumor models. We have studied the antitumor activity of single L19mTNF-α treatment in combination with melphalan and gemcitabine (G) using different administration protocols in two histologically different murine tumor models: WEHI-164 fibrosarcoma and K7M2 osteosarcoma. All responding mice showed significant reduction in myeloid-derived suppressor cells (MDSCs) and an increase in CD4(+) and CD8(+) T cells in the tumor infiltrates, as well as significant reduction in regulatory T cells (Treg) at the level of draining lymph nodes. What is important is that all cured mice rejected tumor challenge up to 1 year after therapy. Targeted delivery of L19mTNF-α synergistically increases the antitumor activity of melphalan and gemcitabine, but optimal administration schedules are required. This study provides information for designing clinical studies using L19mTNF-α in combination with chemotherapeutic drugs.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Modelos Animais de Doenças , Esquema de Medicação , Sinergismo Farmacológico , Memória Imunológica , Depleção Linfocítica , Linfócitos do Interstício Tumoral , Melfalan/administração & dosagem , Camundongos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/mortalidade , Proteínas Recombinantes de Fusão/administração & dosagem , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , GencitabinaRESUMO
The chemopreventive and therapeutic efficacy of the cyclooxygenase (COX) inhibitor ibuprofen (IB) and of sulfasalazine (SASP), a drug that targets the antioxidant xc- system, were exploited in the experimental model of 3-methylcholantrene (3-MCA)-induced mouse sarcoma. The chemopreventive treatments gave unsatisfactory results because administration of IB one day after the 3-MCA injection only slightly delayed the tumor development, whereas SASP dispensed under the same conditions resulted in accelerated tumorigenesis. Similarly, the therapeutic treatment with either drug, administrated daily from the tumor detection, decreased the proliferation rate of tumor cells and increased the survival of treated mice only at a low extent. Remarkably, the combined chemopreventive treatment with IB and therapeutic treatment with SASP displayed a better efficacy, with strong delay of sarcoma growth, reduced tumor size and increased survival of treated mice. The two drugs target not only tumor cells but also tumor-associated macrophages that were dramatically decreased in the tumor infiltrate of mice subjected to the combined treatment. The synergistic effects of the association between a broad anti-inflammatory compound, such as IB, and a redox-directed drug, such as SASP, shed new light in the role of inflammation and of the redox response in chemical tumorigenesis and point to the combined chemopreventive plus therapeutic treatment with IB and SASP as a promising novel approach for antitumor therapy.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Anticarcinógenos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Ibuprofeno/farmacologia , Sarcoma Experimental/prevenção & controle , Sulfassalazina/farmacologia , Adulto , Idoso , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Anticarcinógenos/uso terapêutico , Antioxidantes/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Ibuprofeno/uso terapêutico , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Metilcolantreno , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Sarcoma Experimental/induzido quimicamente , Sarcoma Experimental/imunologia , Sarcoma Experimental/metabolismo , Sulfassalazina/uso terapêutico , Adulto JovemRESUMO
BACKGROUND: Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. We hypothesized that the specific targeting of TNF-alpha antagonists to inflamed tissues could increase drug efficacy and reduce side effects. RESULTS: Using uteroglobin (UG), a potent anti-inflammatory protein, as a scaffold, we prepared a bispecific tetravalent molecule consisting of the extracellular ligand-binding portion of the human TNF-alpha receptor P75 (TNFRII) and the scFv L19. L19 binds to the ED-B containing fibronectin isoform (B-FN), which is expressed only during angiogenesis processes and during tissue remodeling. B-FN has also been demonstrated in the pannus in rheumatoid arthritis. L19-UG-TNFRII is a stable, soluble homodimeric protein that maintains the activities of both moieties: the immuno-reactivity of L19 and the capability of TNFRII to inhibit TNF-alpha. In vivo bio-distribution studies demonstrated that the molecule selectively accumulated on B-FN containing tissues, showing a very fast clearance from the blood but a very long residence time on B-FN containing tissues. Despite the very fast clearance from the blood, this fusion protein was able to significantly improve the severe symptomatology of arthritis in collagen antibody-induced arthritis (CAIA) mouse model. CONCLUSIONS: The recombinant protein described here, able to selectively deliver the TNF-alpha antagonist TNFRII to inflamed tissues, could yield important contributions for the therapy of degenerative inflammatory diseases.
Assuntos
Artrite Experimental/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Fibronectinas/metabolismo , Articulações/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Receptores Tipo II do Fator de Necrose Tumoral/farmacocinética , Proteínas Recombinantes de Fusão/farmacocinética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Células CHO , Cricetinae , Dimerização , Fibronectinas/imunologia , Humanos , Radioisótopos do Iodo/análise , Articulações/efeitos dos fármacos , Articulações/imunologia , Articulações/patologia , Camundongos , Neovascularização Patológica/imunologia , Plasmídeos , Ligação Proteica , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Teratocarcinoma , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Uteroglobina/química , Uteroglobina/genéticaRESUMO
INTRODUCTION: Periostin (PN), a member of the fasciclin family of proteins, is a TGF-ß-induced extracellular matrix protein involved in cell survival, angiogenesis, invasion and metastasis. It is considered a potent angiogenic factor and a marker of tumour progression in many types of human cancer. Many different kinds of cells bind to PN by means of the integrins αvß3 and αvß5, but the periostin epitope recognised by these integrins is not formally demonstrated. The aim of our study was to identify which domain of PN could be involved in cell adhesion and its potential role in tumour growth. METHODS: We generated the monoclonal antibody OC-20 (mAb OC-20) by hybridoma technology. Different PN recombinant fragments were used to characterise the periostin epitope recognised by the mAb OC-20 and to localise a new cell binding site of the protein. A murine model of human melanoma was used in the preclinical in vivo experiments. RESULTS: We formally demonstrate that the periostin epitope recognised by OC-20 is a new binding site for the integrins αvß3 and αvß5, localised in the second FAS1 domain (FAS1-2) of the protein. Moreover the in vivo use of this antibody significantly inhibits tumour growth and angiogenesis. CONCLUSION: Our results show that the FAS1-2 domain of PN plays a role in tumour progression. Moreover this novel antibody may likewise prove to be very useful in clarifying the role of PN in angiogenesis and may contribute to the design of novel anti-angiogenesis drugs.
Assuntos
Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/metabolismo , Integrinas/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Motivos de Aminoácidos , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Experimentais , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estrutura Terciária de Proteína , Neoplasias Cutâneas/patologiaRESUMO
AIMS: Cancer chemoresistance is often due to upregulation of antioxidant systems. Therapeutic targeting of these systems is however hampered by their redundancy. Here, we have performed a functional dissection of the antioxidant systems in different melanoma cases aimed at the identification of the most effective redox active drug. RESULTS: We have identified two crucial antioxidant mechanisms: glutathione (GSH), the major intracellular redox buffer, and the cystine/cysteine cycle, which switches the extracellular redox state from an oxidized to a reduced state. The two mechanisms are independent in melanoma cells and may be substitutes for each other, but targeting both of them is lethal. Exposure to the pro-oxidant compound As(2)O(3) induces an antioxidant response. However, while in these cells the intracellular redox balance remains almost unaffected, a reduced environment is generated extracellularly. GSH depletion by buthioninesulfoximine (BSO), or cystine/cysteine cycle inhibition by (S)-4-carboxyphenylglycine (sCPG), enhanced the sensitivity to As(2)O(3). Remarkably, sCPG also prevented the remodeling of the microenvironment redox state. INNOVATION: We propose that the definition of the prevalent antioxidant system(s) in tumors is crucial for the design of tailored therapies involving redox-directed drugs in association with pro-oxidant drugs. CONCLUSION: In melanoma cells, BSO is the best enhancer of As(2)O(3) sensitivity. However, since the strong remodeling of the microenvironmental redox state caused by As(2)O(3) may promote tumor progression, the concomitant use of cystine/cysteine cycle blockers is recommended.
Assuntos
Antioxidantes/metabolismo , Cisteína/metabolismo , Cistina/metabolismo , Glutationa/metabolismo , Melanoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trióxido de Arsênio , Arsenicais/farmacologia , Western Blotting , Butionina Sulfoximina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Imuno-Histoquímica , Oxirredução/efeitos dos fármacos , Óxidos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
High-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecules, favors tissue regeneration via recruitment and activation of leukocytes and stem cells. Here we demonstrate, in a model of acute sterile muscle injury, that regeneration is accompanied by active reactive oxygen species (ROS) production counterbalanced and overcome by the generation of antioxidant moieties. Mitochondria are initially responsible for ROS formation. However, they undergo rapid disruption with almost complete disappearance. Twenty-four hours after injury, we observed a strong induction of MURF1 and atrogin-1 ubiquitin ligases, key signals in activation of the proteasome system and induction of muscle atrophy. At later time points, ROS generation is maintained by nonmitochondrial sources. The antioxidant response occurs in both regenerating fibers and leukocytes that express high levels of free thiols and antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and thioredoxin. HMGB1, a protein thiol, weakly expressed in healthy muscles, increases during regeneration in parallel with the antioxidant response in both fibers and leukocytes. A reduced environment may be important to maintain HMGB1 bioactivity. Indeed, oxidation abrogates both muscle stem cell migration in response to HMGB1 and their ability to differentiate into myofibers in vitro. We propose that the early antioxidant response in regenerating muscle limits HMGB1 oxidation, thus allowing successful muscle regeneration.