RESUMEN
The surgical resection of primary colorectal cancer is associated with an enhanced risk of liver metastases. Moreover, bacterial translocation or anastomic leakage during resection has been shown to correlate with a poor long-term surgical outcome, suggesting that bacterial products may contribute to the formation of metastases. Driven by these premises, we investigated the role of the bacterial product lipopolysaccharide (LPS) in the generation of liver metastases. Intraperitoneal injection of LPS led to enhanced tumor-cell adhesion to the rat liver as early as 1.5 h post-administration. Furthermore, a rapid loss of the expression of the tight junction protein zonula occludens-1 (ZO-1) was observed, suggesting that LPS disrupts the integrity of the microvasculature. LPS addition to endothelial-macrophage co-cultures damaged endothelial monolayers and caused the formation of intercellular gaps, which was accompanied by increased tumor-cell adhesion. These results suggest that macrophages are involved in the endothelial damage resulting from exposure to LPS. Interestingly, the expression levels of of ZO-1 were not affected by LPS treatment in rats in which liver macrophages had been depleted as well as in rats that had been treated with a reactive oxygen species (ROS) scavenger. In both settings, decreased tumor-cell adhesion was observed. Taken together, our findings indicate that LPS induces ROS release by macrophages, resulting in the damage of the vascular lining of the liver and hence allowing increased tumor-cell adherence. Thus, peri-operative treatments that prevent the activation of macrophages and-as a consequence-limit endothelial damage and tumor-cell adhesion may significantly improve the long-term outcome of cancer patients undergoing surgical tumor resection.
RESUMEN
Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.
Asunto(s)
Anticuerpos Biespecíficos/fisiología , Antígenos CD/fisiología , Autofagia/inmunología , Marcación de Gen/métodos , Neutrófilos/inmunología , Neutrófilos/patología , Receptores Fc/fisiología , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis/inmunología , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Humanos , Inmunoglobulina A/fisiología , Neutrófilos/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismoRESUMEN
OBJECTIVE: Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) are shown to be produced and released during surgery, the effects of ROS on the liver vascular lining and tumour cell adhesion were investigated. METHODS: Human endothelial cell monolayers (human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells of the lung (HMEC-1s)) were exposed to ROS production, after which electrical impedance, cellular integrity and tumour cell adhesion were investigated. Furthermore, surgery-induced tumour cell adhesion as well as the role of ROS and liver macrophages (Kupffer cells) in this process were studied in vivo. RESULTS: Production of ROS decreased cellular impedance of endothelial monolayers dramatically. Moreover, formation of intercellular gaps in endothelial monolayers was observed, exposing subendothelial extracellular matrix (ECM) on which colon carcinoma cells adhered via integrin molecules. Endothelial damage was, however, prevented in the presence of ROS-scavenging enzymes. Additionally, surgery induced downregulation of both rat and human liver tight junction molecules. Treatment of rats with the ROS scavenger edaravone prevented surgery-induced tumour cell adhesion and downregulation of tight junction proteins in the liver. Interestingly, depletion of Kupffer cells prior to surgery significantly reduced the numbers of adhered tumour cells and prevented disruption of expression of tight junction proteins. CONCLUSIONS: In this study it is shown that surgery-induced ROS production by macrophages damages the vascular lining by downregulating tight junction proteins. This leads to exposure of ECM, to which circulating tumour cells bind. In light of this, perioperative therapeutic intervention, preventing surgery-induced inflammatory reactions, may reduce the risk of developing liver metastases, thereby improving the clinical outcome of patients with colorectal cancer.
Asunto(s)
Carcinoma/secundario , Colectomía/efectos adversos , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/secundario , Neoplasias Experimentales/patología , Especies Reactivas de Oxígeno/farmacología , Animales , Biopsia , Carcinoma/etiología , Carcinoma/metabolismo , Agregación Celular , Línea Celular Tumoral , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Neoplasias Experimentales/etiología , Neoplasias Experimentales/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.
Asunto(s)
Anomalías Inducidas por Medicamentos/enzimología , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/metabolismo , Teratógenos/toxicidad , Tiocarbamatos/toxicidad , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismo , Anomalías Inducidas por Medicamentos/embriología , Animales , Inhibidores Enzimáticos/toxicidad , Técnicas de Silenciamiento del Gen/métodos , Proteína-Lisina 6-Oxidasa/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
UNLABELLED: Currently, an operation is the only curative option for patients with colorectal cancer. Unfortunately, many patients will develop liver metastases even after successful resection of the primary tumor. Removal of primary colorectal carcinoma may paradoxically increase the risk of metastases development, because accumulating evidence suggests that surgical trauma can stimulate tumor growth. In the present study, we investigated the effects of abdominal trauma on liver metastases development. Surgical trauma dramatically increased adhesion of tumor cells in the liver, leading to enhanced outgrowth of metastases. Endothelial stress was observed rapidly after an operation, suggesting that abdominal trauma resulted in impairment of blood vessel integrity. Tumor cells preferentially adhered to extracellular matrix (ECM). Furthermore, preincubation of tumor cells with anti-alpha2 integrin antibodies completely reverted operation-induced augmentation of CC531s adhesion and liver metastases outgrowth. As such, we postulate that blood vessel integrity in the liver is compromised after abdominal trauma, resulting in enhanced ECM exposure, which enables tumor cell adhesion and metastases outgrowth. CONCLUSION: Perioperative treatments that either aim to reduce endothelial stress or block the interaction between tumor cells and ECM represent promising new therapeutic strategies for the prevention of liver metastases development after resection of the primary tumor.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Integrina alfa2/inmunología , Neoplasias Hepáticas/secundario , Metástasis de la Neoplasia/prevención & control , Animales , Línea Celular , Citometría de Flujo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Microscopía Confocal , Metástasis de la Neoplasia/patología , RatasRESUMEN
The study of early events in dendritic cell (DC) differentiation is hampered by the lack of homogeneous primary cell systems that allow the study of cytokine-driven, transitional DC differentiation steps. The CD34(+) acute myeloid leukemia cell line MUTZ-3 displays a unique ability to differentiate into interstitial DC (IDC) and Langerhans cells (LC) in a cytokine-dependent manner. Phenotypic characterization revealed MUTZ-3 to consist of three distinct subpopulations. Small CD34(+)CD14(-)CD11b(-) progenitors constitute the proliferative compartment of the cell line with the ability to differentiate through a CD34(-)CD14(-)CD11b(+) stage to ultimately give rise to a morphologically large, nonproliferating CD14(+)CD11b(hi) progeny. These CD14(+)CD11b(hi) cells were identified as common, immediate myeloid DC precursors with the ability to differentiate into LC and IDC, exhibiting characteristic and mutually exclusive expression of Langerin and DC-specific ICAM-grabbing nonintegrin, respectively. The identity of the MUTZ-3-derived LC subset was confirmed further by the presence of Birbeck granules. We conclude that the MUTZ-3 cell line provides a ready and continuous supply of common myeloid precursors, which should facilitate further study of the ontogeny of myeloid DC lineages.
Asunto(s)
Antígenos CD34/inmunología , Antígeno CD11b/inmunología , Diferenciación Celular/inmunología , Células de Langerhans/inmunología , Receptores de Lipopolisacáridos/inmunología , Modelos Inmunológicos , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos CD34/metabolismo , Antígeno CD11b/metabolismo , Línea Celular Tumoral , Proliferación Celular , Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Células de Langerhans/metabolismo , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/inmunología , Receptores de Lipopolisacáridos/metabolismo , Lectinas de Unión a Manosa/biosíntesis , Lectinas de Unión a Manosa/inmunología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismoRESUMEN
The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.
Asunto(s)
Microscopía por Crioelectrón/métodos , Microbiología/instrumentación , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/ultraestructura , Tuberculosis Pulmonar/microbiología , Antígenos Bacterianos/análisis , Células Cultivadas , Humanos , Lepra/microbiología , Lípidos/análisis , Microscopía Fluorescente/métodos , Mycobacterium leprae/química , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/ultraestructura , Mycobacterium tuberculosis/químicaRESUMEN
The maturation of dendritic cells is accompanied by the redistribution of major histocompatibility complex (MHC) class II molecules from the lysosomal MHC class II compartment to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CD1 molecules that mediate presentation of lipid antigens. Herein, we show that in human monocyte-derived dendritic cells, unlike MHC class II, the steady-state distribution of lysosomal CD1b and CD1c isoforms was unperturbed in response to lipopolysaccharide-induced maturation. However, the lysosomes in these cells underwent a dramatic reorganization into electron dense tubules with altered lysosomal protein composition. These structures matured into novel and morphologically unique compartments, here termed mature dendritic cell lysosomes (MDL). Furthermore, we show that upon activation mature dendritic cells do not lose their ability of efficient clathrin-mediated endocytosis as demonstrated for CD1b and transferrin receptor molecules. Thus, the constitutive endocytosis of CD1b molecules and the differential sorting of MHC class II from lysosomes separate peptide- and lipid antigen-presenting molecules during dendritic cell maturation.