RESUMO
Eosinophilia is a feature of multiple conditions, both hematologic and non-hematologic, and may be associated with organ damage. The pathogenesis of eosinophilia can follow two distinct pathways. Primary eosinophilia is caused by a cell-intrinsic mechanism originating from clonal expansion of eosinophils through acquisition of a somatic mutation, such as FIP1L1-PDGFRA. In recent years, great progress has been made in the field of pathogenesis and molecularly targeted therapy of neoplastic eosinophilia. The diagnostic procedure should include, among other things, morphologic analysis of blood and bone marrow samples, cytogenetics and fluorescence in situ-hybridization tests to detect evidence of an acute or chronic myeloid or lymphoid disorder. Secondary eosinophilia follows a cell-extrinsic mechanism as a response to exogenous cytokines. In most clinical cases, peripheral blood eosinophilia is reactive and typically associated with non-hematological disorders such as infections, allergic conditions, connective tissue disorders, vasculitis, malignancy, or endocrinopathies. Nonetheless, the cause of most cases of hypereosinophilic syndrome remains unknown. In this article, we present a short review focused on differential diagnosis of eosinophilia and eosinophilic disorders. The diagnosis of eosinophilia is a challenge for physicians; thus this review may be useful in clinical practice.
RESUMO
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
Processing of the 'CaaX' motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these 'CaaX' proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper 'CaaX' processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.
Assuntos
Membrana Celular/metabolismo , Endopeptidases/metabolismo , Endopeptidases/fisiologia , Genes ras/fisiologia , Membrana Celular/química , Células HEK293 , Células HeLa , Humanos , Isoformas de Proteínas/metabolismo , Proto-Oncogene MasRESUMO
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
meso-Tetra(N-methyl-4-pyridyl) porphine tetra tosylate (TMP) is a photosensitizer that can be used in photodynamic therapy (PDT) to induce cell death through generation of reactive oxygen species in targeted tumor cells. However, TMP is highly hydrophilic, and therefore, its ability to accumulate intracellularly is limited. In this study, a strategy to improve TMP uptake into cells has been investigated by encapsulating the compound in a hydrogel-based chitosan/alginate nanoparticle formulation. Nanoparticles of 560 nm in diameter entrapping 9.1 µg of TMP per mg of formulation were produced and examined in cell-based assays. These particles were endocytosed into human colorectal carcinoma HCT116 cells and elicited a more potent photocytotoxic effect than free drug. Antibodies targeting death receptor 5 (DR5), a cell surface apoptosis-inducing receptor up-regulated in various types of cancer and found on HCT116 cells, were then conjugated onto the particles. The conjugated antibodies further enhanced uptake and cytotoxic potency of the nanoparticle. Taken together, these results show that antibody-conjugated chitosan/alginate nanoparticles significantly enhanced the therapeutic effectiveness of entrapped TMP. This novel approach provides a strategy for providing targeted site-specific delivery of TMP and other photosensitizer drugs to treat colorectal tumors using PDT.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/uso terapêutico , Alginatos , Anticorpos/imunologia , Transporte Biológico , Caspase 8/metabolismo , Linhagem Celular Tumoral , Quitosana/imunologia , Ácido Glucurônico/imunologia , Ácidos Hexurônicos/imunologia , Humanos , Nanopartículas , Fotoquimioterapia , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologiaRESUMO
The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.
Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Substituição de Aminoácidos , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Salmonella enteritidis/metabolismo , Adesinas Bacterianas/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella enteritidis/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.
Assuntos
Clatrina/metabolismo , Endopeptidases/metabolismo , Receptores ErbB/metabolismo , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Endocitose , Endopeptidases/genética , Células HeLa , Humanos , TransfecçãoRESUMO
Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.
Assuntos
Lipocalinas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cricetulus , Relação Dose-Resposta a Droga , Mapeamento de Epitopos , Feminino , Células HT29 , Fator de Crescimento de Hepatócito/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Lipocalinas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas/uso terapêutico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.
Assuntos
Movimento Celular/fisiologia , Endopeptidases/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Interleucina-8/metabolismo , Transporte Proteico , Proteínas rho de Ligação ao GTP/análiseRESUMO
Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.