RESUMO
Radiotherapy (RT) treatment is an important strategy for the management of non-small cell lung cancer (NSCLC). Local recurrence amongst patients with late-stage NSCLC remains a challenge. The loss of PTEN has been associated with radio-resistance. This study aimed to examine the efficacy of RT combined with ataxia telangiectasia-mutated Rad3-related (ATR) inhibition using Ceralasertib in phosphatase and tensin homolog (PTEN)-depleted NSCLC cells and to assess early inflammatory responses indicative of radiation pneumonitis (RP) after combined-modality treatment. Small hairpin RNA (shRNA) transfections were used to generate H460 and A549 PTEN-depleted models. Ceralasertib was evaluated as a single agent and in combination with RT in vitro and in vivo. Histological staining was used to assess immune cell infiltration in pneumonitis-prone C3H/NeJ mice. Here, we report that the inhibition of ATR in combination with RT caused a significant reduction in PTEN-depleted NSCLC cells, with delayed DNA repair and reduced cell viability, as shown by an increase in cells in Sub G1. Combination treatment in vivo significantly inhibited H460 PTEN-depleted tumour growth in comparison to H460 non-targeting PTEN-expressing (NT) cell-line-derived xenografts (CDXs). Additionally, there was no significant increase in infiltrating macrophages or neutrophils except at 4 weeks, whereby combination treatment significantly increased macrophage levels relative to RT alone. Overall, our study demonstrates that ceralasertib and RT combined preferentially sensitises PTEN-depleted NSCLC models in vitro and in vivo, with no impact on early inflammatory response indicative of RP. These findings provide a rationale for evaluating ATR inhibition in combination with RT in NSCLC patients with PTEN mutations.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , PTEN Fosfo-Hidrolase , Pirimidinas , Tolerância a Radiação , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Animais , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Camundongos , Tolerância a Radiação/efeitos dos fármacos , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Linhagem Celular Tumoral , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Reparo do DNA/efeitos dos fármacos , Indóis , Morfolinas , SulfonamidasRESUMO
Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.
Assuntos
Pneumonia , Síndrome do Desconforto Respiratório , Animais , Líquido da Lavagem Broncoalveolar , Catepsinas , Modelos Animais de Doenças , Humanos , Pulmão/patologia , CamundongosRESUMO
OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.
Assuntos
Biomarcadores Tumorais , Neoplasias do Colo , Humanos , Biomarcadores Tumorais/genética , Células Estromais/patologia , Recidiva Local de Neoplasia/prevenção & controle , Recidiva Local de Neoplasia/patologia , Neoplasias do Colo/patologia , PrognósticoRESUMO
BACKGROUND: Elevated levels of the cysteine protease cathepsin S (CatS) are associated with chronic mucoobstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). We have previously demonstrated that prophylactic treatment with a CatS inhibitor from birth reduces inflammation, mucus plugging, and lung tissue damage in juvenile ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice with chronic inflammatory mucoobstructive lung disease. In this study, we build upon this work to examine the effects of therapeutic intervention with a CatS inhibitor in adult ßENaC-Tg mice with established disease. METHODS: ßENaC-Tg mice and wild-type (WT) littermates were treated with a CatS inhibitor from 4 to 6 weeks of age, and CatS-/- ßENaC-Tg mice were analysed at 6 weeks of age. Bronchoalveolar lavage (BAL) fluid inflammatory cell counts were quantified, and lung tissue destruction and mucus obstruction were analysed histologically. RESULTS: At 6 weeks of age, ßENaC-Tg mice developed significant airway inflammation, lung tissue damage, and mucus plugging when compared to WT mice. CatS-/- ßENaC-Tg mice and ßENaC-Tg mice receiving inhibitor had significantly reduced airway mononuclear and polymorphonuclear (PMN) cell counts as well as mucus plugging. However, in contrast to CatS-/- ßENaC-Tg mice, therapeutic inhibition of CatS in ßENaC-Tg mice had no effect on established emphysema-like lung tissue damage. CONCLUSIONS: These results suggest that while early CatS targeting may be required to prevent the onset and progression of lung tissue damage, therapeutic CatS targeting effectively inhibited airway inflammation and mucus obstruction. These results indicate the important role CatS may play in the pathogenesis and progression of mucoobstructive lung disease.
Assuntos
Catepsinas/antagonistas & inibidores , Fibrose Cística , Canais Epiteliais de Sódio , Animais , Fibrose Cística/patologia , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/patologia , Pulmão/patologia , Camundongos , Camundongos Transgênicos , MucoRESUMO
Rationale: Acute respiratory distress syndrome is defined by the presence of systemic hypoxia and consequent on disordered neutrophilic inflammation. Local mechanisms limiting the duration and magnitude of this neutrophilic response remain poorly understood. Objectives: To test the hypothesis that during acute lung inflammation tissue production of proresolution type 2 cytokines (IL-4 and IL-13) dampens the proinflammatory effects of hypoxia through suppression of HIF-1α (hypoxia-inducible factor-1α)-mediated neutrophil adaptation, resulting in resolution of lung injury. Methods: Neutrophil activation of IL4Ra (IL-4 receptor α) signaling pathways was explored ex vivo in human acute respiratory distress syndrome patient samples, in vitro after the culture of human peripheral blood neutrophils with recombinant IL-4 under conditions of hypoxia, and in vivo through the study of IL4Ra-deficient neutrophils in competitive chimera models and wild-type mice treated with IL-4. Measurements and Main Results: IL-4 was elevated in human BAL from patients with acute respiratory distress syndrome, and its receptor was identified on patient blood neutrophils. Treatment of human neutrophils with IL-4 suppressed HIF-1α-dependent hypoxic survival and limited proinflammatory transcriptional responses. Increased neutrophil apoptosis in hypoxia, also observed with IL-13, required active STAT signaling, and was dependent on expression of the oxygen-sensing prolyl hydroxylase PHD2. In vivo, IL-4Ra-deficient neutrophils had a survival advantage within a hypoxic inflamed niche; in contrast, inflamed lung treatment with IL-4 accelerated resolution through increased neutrophil apoptosis. Conclusions: We describe an important interaction whereby IL4Rα-dependent type 2 cytokine signaling can directly inhibit hypoxic neutrophil survival in tissues and promote resolution of neutrophil-mediated acute lung injury.
Assuntos
Lesão Pulmonar Aguda/imunologia , Subunidade alfa de Receptor de Interleucina-4/imunologia , Interleucina-4/imunologia , Neutrófilos/imunologia , Receptores de Superfície Celular/imunologia , Síndrome do Desconforto Respiratório/imunologia , Lesão Pulmonar Aguda/metabolismo , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Receptores de Superfície Celular/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de SinaisRESUMO
Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS-/-) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of ßENaC-Tg mice compared with wild-type (WT) littermates. CatS-/-ßENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with ßENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of ßENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in ßENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target.
Assuntos
Catepsinas/metabolismo , Fibrose Cística/metabolismo , Muco/metabolismo , Pneumonia/metabolismo , Receptor PAR-2/metabolismo , Obstrução das Vias Respiratórias/metabolismo , Animais , Catepsinas/genética , Modelos Animais de Doenças , Canais Epiteliais de Sódio/genética , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/etiologiaRESUMO
Eppin is a serine protease inhibitor expressed in male reproductive tissues.The aim of this study was to investigate the localisation and regulation of eppin expression in myeloid and epithelial cell lines, and explore its potential role as a multifunctional host defence protein.Using immunohistochemistry and Western blotting, eppin was detected in the lungs of patients with acute respiratory distress syndrome and cystic fibrosis lung disease. Expression of eppin in monocytic cells was unaffected by stimulation with Toll-like receptor agonists, cytokines and hormone receptor agonists. However, upregulated expression and secretion of eppin was observed following treatment of monocytes with epidermal growth factor. Incubation of recombinant eppin with monocytic cells resulted in significant inhibition of lipopolysaccharide-induced chemokine production. Furthermore, eppin inhibited lipopolysaccharide-induced NF-κB activation by a mechanism which involved accumulation of phosphorylated IκBα. In an in vivo model of lung inflammation induced by lipopolysaccharide, eppin administration resulted in decreased recruitment of neutrophils to the lung with a concomitant reduction in the levels of the neutrophil chemokine macrophage inflammatory protein-2.Overall, these results suggest a role for eppin outside of the reproductive tract and that eppin may have a role in the innate immune response in the lung.
Assuntos
Fibrose Cística/metabolismo , Citocinas/metabolismo , Pulmão/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular Tumoral , Humanos , Imunidade Inata , Masculino , Síndrome do Desconforto Respiratório/genética , Transdução de Sinais , Escarro/química , Receptores Toll-Like/metabolismoRESUMO
Members of the whey acidic protein (WAP) or WAP four-disulfide-core (WFDC) family of proteins are a relatively under-explored family of low molecular weight proteins. The two most prominent WFDC proteins, secretory leukocyte protease inhibitor (SLPI) and elafin (or the precursor, trappin-2), have been shown to possess multiple functions including anti-protease, anti-bacterial, anti-viral and anti-inflammatory properties. It is therefore of no surprise that both SLPI and elafin/trappin-2 have been developed as potential therapeutics. Given the abundance of SLPI and elafin/trappin-2 in the human lung, most work in the area of WFDC research has focused on the role of WFDC proteins in protecting the lung from proteolytic attack. In this review, we will outline the current evidence regarding the expanding role of WFDC protein function with a focus on WFDC activity in lung disease as well as emerging data regarding the function of some of the more recently described WFDC proteins.
Assuntos
Pneumopatias/fisiopatologia , Proteínas do Leite/metabolismo , Fenômenos Fisiológicos Respiratórios , Humanos , Pneumopatias/prevenção & controle , Proteínas do Leite/classificação , Proteínas/metabolismo , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
RATIONALE: IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Although IL-17A is the archetypal cytokine of T-helper 17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown. OBJECTIVES: To identify the cellular source and the role of IL-17A in the early phase of lung injury. METHODS: Lung injury was induced in wild-type (C57BL/6) and IL-17 knockout (KO) mice with aerosolized LPS (100 µg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was performed by flow cytometry. MEASUREMENTS AND MAIN RESULTS: A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared with wild-type mice. The majority of RORγt(+) cells in the mouse lung were the recently identified group 3 innate lymphoid cells (ILC3s). Detailed characterization revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in recombinase-activating gene 2 KO mice, which lack T cells but retain innate lymphoid cells. No amelioration of pathology was observed in the recombinase-activating gene 2 KO mice. CONCLUSIONS: IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3s. Modulation of the activity of pILC3s may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.
Assuntos
Interleucina-17/biossíntese , Linfócitos/patologia , Síndrome do Desconforto Respiratório/patologia , Animais , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Pulmão/patologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Síndrome do Desconforto Respiratório/metabolismoRESUMO
Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden.
Assuntos
Anti-Inflamatórios/farmacologia , Elafina/farmacologia , Pulmão/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Líquido da Lavagem Broncoalveolar/química , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Elafina/química , Elafina/genética , Fibronectinas/antagonistas & inibidores , Fibronectinas/metabolismo , Expressão Gênica , Humanos , Cinética , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Dados de Sequência Molecular , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Inibidores de Proteases/química , Engenharia de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transglutaminases/antagonistas & inibidores , Transglutaminases/metabolismoRESUMO
INTRODUCTION: Secretory leucocyte protease inhibitor and elafin are members of the whey acidic protein (WAP), or WAP four disulfide-core (WFDC), family of proteins and have multiple contributions to innate defence including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). This study aimed to explore potential activities of WFDC12, a previously uncharacterised WFDC protein expressed in the lung. METHODS: Recombinant expression and purification of WFDC12 were optimised in Escherichia coli. Antiprotease, antibacterial and immunomodulatory activities of recombinant WFDC12 were evaluated and levels of endogenous WFDC12 protein were characterised by immunostaining and ELISA. RESULTS: Recombinant WFDC12 inhibited cathepsin G, but not elastase or proteinase-3 activity. Monocytic cells pretreated with recombinant WFDC12 before LPS stimulation produced significantly lower levels of the pro-inflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared with cells stimulated with LPS alone. Recombinant WFDC12 became conjugated to fibronectin in a transglutaminase-mediated reaction and retained antiprotease activity. In vivo WFDC12 expression was confirmed by immunostaining of human lung tissue sections. WFDC12 levels in human bronchoalveolar lavage fluid from healthy and lung-injured patients were quantitatively compared, showing WFDC12 to be elevated in both patients with acute respiratory distress syndrome and healthy subjects treated with LPS, relative to healthy controls. CONCLUSIONS: Together, these results suggest a role for this lesser known WFDC protein in the regulation of lung inflammation.
Assuntos
Pulmão/metabolismo , Monócitos/efeitos dos fármacos , Proteínas/farmacologia , Serina Endopeptidases/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/química , Humanos , Lipopolissacarídeos , Pulmão/patologia , Testes de Sensibilidade Microbiana , Monócitos/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Técnicas de Cultura de TecidosRESUMO
Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.
Assuntos
Polipose Adenomatosa do Colo/terapia , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/administração & dosagem , Nanopartículas/administração & dosagem , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Camptotecina/farmacologia , Linhagem Celular Tumoral , Feminino , Células HCT116 , Células HT29 , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Nanomedicina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent murine studies have demonstrated that tumor-associated macrophages in the tumor microenvironment are a key source of the pro-tumorigenic cysteine protease, cathepsin S. We now show in a syngeneic colorectal carcinoma murine model that both tumor and tumor-associated cells contribute cathepsin S to promote neovascularization and tumor growth. Cathepsin S depleted and control colorectal MC38 tumor cell lines were propagated in both wild type C57Bl/6 and cathepsin S null mice to provide stratified depletion of the protease from either the tumor, tumor-associated host cells, or both. Parallel analysis of these conditions showed that deletion of cathepsin S inhibited tumor growth and development, and revealed a clear contribution of both tumor and tumor-associated cell derived cathepsin S. The most significant impact on tumor development was obtained when the protease was depleted from both sources. Further characterization revealed that the loss of cathepsin S led to impaired tumor vascularization, which was complemented by a reduction in proliferation and increased apoptosis, consistent with reduced tumor growth. Analysis of cell types showed that in addition to the tumor cells, tumor-associated macrophages and endothelial cells can produce cathepsin S within the microenvironment. Taken together, these findings clearly highlight a manner by which tumor-associated cells can positively contribute to developing tumors and highlight cathepsin S as a therapeutic target in cancer.
Assuntos
Carcinoma Pulmonar de Lewis/patologia , Catepsinas/fisiologia , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Melanoma Experimental/patologia , Neovascularização Patológica , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/genética , Adesão Celular , Ciclo Celular , Células Cultivadas , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Macrófagos/citologia , Macrófagos/metabolismo , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microambiente TumoralRESUMO
Secretory leucoprotease inhibitor (SLPI) has multifaceted functions, including inhibition of protease activity, antimicrobial functions, and anti-inflammatory properties. In this study, we show that SLPI plays a role in controlling pulmonary Pseudomonas aeruginosa infection. Mice lacking SLPI were highly susceptible to P. aeruginosa infection, however there was no difference in bacterial burden. Utilising a model of P. aeruginosa LPS-induced lung inflammation, human recombinant SLPI (hrSLPI) administered intraperitoneally suppressed the recruitment of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and resulted in reduced BALF and serum levels of inflammatory cytokines and chemokines. This anti-inflammatory effect of hrSLPI was similarly demonstrated in a systemic inflammation model induced by intraperitoneal injection of LPS from various bacteria or lipoteichoic acid, highlighting the broad anti-inflammatory properties of hrSLPI. Moreover, in bone-marrow-derived macrophages, hrSLPI reduced LPS-induced phosphorylation of p-IkB-α, p-IKK-α/ß, p-P38, demonstrating that the anti-inflammatory effect of hrSLPI was due to the inhibition of the NFκB and MAPK pathways. In conclusion, administration of hrSLPI attenuates excessive inflammatory responses and is therefore, a promising strategy to target inflammatory diseases such as acute respiratory distress syndrome or sepsis and could potentially be used to augment antibiotic treatment.
Assuntos
Inflamação , Infecções por Pseudomonas , Inibidor Secretado de Peptidases Leucocitárias , Animais , Humanos , Camundongos , Inflamação/metabolismo , Inflamação/microbiologia , Lipopolissacarídeos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/terapia , Inibidor Secretado de Peptidases Leucocitárias/administração & dosagem , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Proteínas Recombinantes/administração & dosagemRESUMO
PURPOSE: The aim of this study was to define the dose and dose-volume relationship of radiation-induced pulmonary toxicities occurring in and out-of-field in mouse models of early inflammatory and late fibrotic response. MATERIALS AND METHODS: Early radiation-induced inflammation and fibrosis were investigated in C3H/NeJ and C57BL/6J mice, respectively. Animals were irradiated with 20 Gy delivered to the upper region of the right lung as a single fraction or as 3 consecutive fractions using the Small Animal Radiation Research Platform (Xstrahl Inc, Camberley, UK). Cone beam computed tomography was performed for image guidance before irradiation and to monitor late toxicity. Histologic sections were examined for neutrophil and macrophage infiltration as markers of early inflammatory response and type I collagen staining as a marker of late-occurring fibrosis. Correlation was evaluated with the dose-volume histogram parameters calculated for individual mice and changes in the observed cone beam computed tomography values. RESULTS: Mean lung dose and the volume receiving over 10 Gy (V10) showed significant correlation with late responses for single and fractionated exposures in directly targeted volumes. Responses observed outside the target volume were attributed to nontargeted effects and showed no dependence on either mean lung dose or V10. CONCLUSIONS: Quantitative assessment of normal tissue response closely correlates early and late pulmonary response with clinical parameters, demonstrating this approach as a potential tool to facilitate clinical translation of preclinical studies. Out-of-field effects were observed but did not correlate with dosimetric parameters, suggesting that nontargeted effects may have a role in driving toxicities outside the treatment field.
Assuntos
Pulmão/efeitos da radiação , Pneumonite por Radiação/patologia , Radioterapia Guiada por Imagem , Animais , Contagem de Células , Colágeno Tipo I/análise , Tomografia Computadorizada de Feixe Cônico , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Pulmão/diagnóstico por imagem , Pulmão/patologia , Macrófagos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neutrófilos , Lesões por Radiação/diagnóstico por imagem , Lesões por Radiação/etiologia , Lesões por Radiação/patologia , Pneumonite por Radiação/diagnóstico por imagem , Dosagem RadioterapêuticaRESUMO
Klebsiella pneumoniae is a foremost gram-negative pathogen that can induce life-threatening nosocomial pulmonary infections. Although it can be phagocytosed successfully by lung resident macrophages, this pathogen remains viable within vacuolar compartments, resulting in chronic infection and limiting therapeutic treatment with antibiotics. In this study, we aimed to generate and evaluate a cell-penetrant antibiotic poly(lactide-co-glycolide) (PLGA)-based formulation that could successfully treat intracellular K. pneumoniae infection. Screening of formulation conditions allowed the generation of high drug loaded nanoparticles through a water-in-oil-in-water approach. We demonstrated the therapeutic usefulness of these gentamicin-loaded nanoparticles (GNPs), showing their ability to improve survival and provide extended prophylactic protection towards K. pneumoniae using a Galleria mellonella infection model. We subsequently showed that the GNPs could be phagocytosed by K. pneumoniae infected macrophages, and significantly reduce the viability of the intracellular bacteria without further stimulation of pro-inflammatory or pro-apoptotic effects on the macrophages. Taken together, these results clearly show the potential to use antibiotic loaded NPs to treat intracellular K. pneumoniae infection, reducing bacterial viability without concomitant stimulation of inflammatory or pyroptotic pathways in the treated cells.
Assuntos
Antibacterianos/administração & dosagem , Gentamicinas/administração & dosagem , Infecções por Klebsiella/tratamento farmacológico , Nanopartículas , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Portadores de Fármacos/química , Gentamicinas/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Macrófagos/metabolismo , Mariposas/microbiologia , Fagocitose/fisiologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/químicaRESUMO
BACKGROUND AND PURPOSE: To evaluate the impact of ATR inhibition using AZD6738 in combination with radiotherapy on the response of non-small cell lung cancer (NSCLC) tumour models and a murine model of radiation induced fibrosis. MATERIALS AND METHODS: AZD6738 was evaluated as a monotherapy and in combination with radiation in vitro and in vivo using A549 and H460 NSCLC models. Radiation induced pulmonary fibrosis was evaluated by cone beam computed tomography (CBCT) and histological staining. RESULTS: AZD6738 specifically inhibits ATR kinase and enhanced radiobiological response in NSCLC models but not in human bronchial epithelial cells (HBECs) in vitro. Significant tumour growth delay was observed in cell line derived xenografts (CDXs) of H460 cells (p<0.05) which were less significant in A549 cells. Combination of AZD6738 with radiotherapy showed no significant change in lung tissue density by CBCT (p>0.5) and histological scoring of radiation induced fibrosis (p>0.5). CONCLUSION: Inhibition of ATR with AZD6738 in combination with radiotherapy increases tumour growth delay without observable augmentation of late radiation induced toxicity further underpinning translation towards clinical evaluation in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/radioterapia , Pirimidinas/farmacologia , Sulfóxidos/farmacologia , Índice Terapêutico , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Linhagem Celular Tumoral , Tomografia Computadorizada de Feixe Cônico , Feminino , Humanos , Indóis , Camundongos , Camundongos Endogâmicos C57BL , Morfolinas , Sulfonamidas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA depletion of CatS resulted in reduced CCL2 expression. This regulation is transcriptionally mediated, as evident from RT-PCR analysis and CCL2 promoter studies. We revealed that CatS regulation of CCL2 is modulated through CD74 (also known as the invariant chain), a known substrate of CatS and a mediator of NFkB activity. Furthermore, CatS and CCL2 show a strong clinical correlation in brain, breast and colon tumours. In summary, these results highlight a novel mechanism by which CatS controls CCL2, which may present a useful pharmacodynamic marker for CatS inhibition.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Catepsinas/genética , Quimiocina CCL2/genética , Antígenos de Histocompatibilidade Classe II/genética , Ativação Transcricional , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Western Blotting , Catepsinas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Quimiocina CCL2/metabolismo , Células HEK293 , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Sepsis is the most frequent cause of death in hospitalized patients, and severe sepsis is a leading contributory factor to acute respiratory distress syndrome (ARDS). At present, there is no effective treatment for these conditions, and care is primarily supportive. Murine sialic acid-binding immunoglobulin-like lectin-E (Siglec-E) and its human orthologs Siglec-7 and Siglec-9 are immunomodulatory receptors found predominantly on hematopoietic cells. These receptors are important negative regulators of acute inflammatory responses and are potential targets for the treatment of sepsis and ARDS. We describe a Siglec-targeting platform consisting of poly(lactic-co-glycolic acid) nanoparticles decorated with a natural Siglec ligand, di(α2â8) N-acetylneuraminic acid (α2,8 NANA-NP). This nanoparticle induced enhanced oligomerization of the murine Siglec-E receptor on the surface of macrophages, unlike the free α2,8 NANA ligand. Furthermore, treatment of murine macrophages with these nanoparticles blocked the production of lipopolysaccharide-induced inflammatory cytokines in a Siglec-E-dependent manner. The nanoparticles were also therapeutically beneficial in vivo in both systemic and pulmonary murine models replicating inflammatory features of sepsis and ARDS. Moreover, we confirmed the anti-inflammatory effect of these nanoparticles on human monocytes and macrophages in vitro and in a human ex vivo lung perfusion (EVLP) model of lung injury. We also established that interleukin-10 (IL-10) induced Siglec-E expression and α2,8 NANA-NP further augmented the expression of IL-10. Indeed, the effectiveness of the nanoparticle depended on IL-10. Collectively, these results demonstrated a therapeutic effect of targeting Siglec receptors with a nanoparticle-based platform under inflammatory conditions.
Assuntos
Inflamação/prevenção & controle , Ácido N-Acetilneuramínico/química , Nanopartículas , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/efeitos dos fármacos , Animais , Humanos , Interleucina-10/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Regulação para CimaRESUMO
Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality.