ABSTRACT
Recently, neutrophil extracellular traps (NETs), three-dimensional structures formed of neutrophil enzymes such as neutrophil elastase (NE) and nuclear components (DNA), have been associated with progression in different types of cancer. However, data remain scarce in breast cancer. Thus, the aim of this study was to associate NETs with clinical stages of breast cancer. A prospective analysis was performed in 45 plasma samples of female patients with newly diagnosed breast cancer. NE-DNA complexes were evaluated by ELISA. Optical density was dichotomized at the median for comparisons (low and high levels of NE-DNA). The most frequent clinical stage was localized (n = 28, 62%) followed by regional (n = 13, 29%) and distant (n = 4, 9%). Higher levels of NE-DNA complexes were observed in regional and distant stages compared to localized disease (68% vs 32%, p = 0.034). No differences were observed when comparing other clinical characteristics between both groups. We demonstrated that the levels of NETs increase in proportion to the stage of the disease, observing higher levels of NE-DNA complexes in regional and metastatic disease, which coincides with the proposed mechanism by which cancer progression and metastasis might result from the formation of NETs.
Subject(s)
Breast Neoplasms/immunology , Breast Neoplasms/pathology , Extracellular Traps/immunology , Adult , Aged , Aged, 80 and over , Disease Progression , Female , Humans , Middle Aged , Neoplasm StagingABSTRACT
BACKGROUND: In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). RESULTS: The kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ' were similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. CONCLUSION: A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.
Subject(s)
Endothelial Cells/metabolism , Fibrinogens, Abnormal/metabolism , Peptide Fragments/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Thrombosis , Urokinase-Type Plasminogen Activator/metabolism , Blood Coagulation , Cell Line , Fibrin/chemistry , Fibrinogen/chemistry , Fibrinolysis/physiology , Humans , Thrombin/metabolism , Thrombosis/metabolismABSTRACT
BACKGROUND: Ischemic heart disease, cerebrovascular accident, and venous thromboembolism have the presence of a thrombotic event in common and represent the most common causes of death within the population. OBJECTIVE: Since Schiff base copper(II) complexes are able to interact with polyphosphates (PolyP), a procoagulant and potentially prothrombotic platelet agent, we investigated the antiplatelet aggregating properties of two novel tridentate Schiff base ligands and their corresponding copper( II) complexes. METHODS: The Schiff base ligands (L1) and (L2), as well as their corresponding copper(II) complexes (C1) and (C2), were synthesized and characterized by chemical analysis, X-ray diffraction, mass spectrometry, and UV-Visible, IR and far IR spectroscopy. In addition, EPR studies were carried out for (C1) and (C2), while (L1) and (L2) were further analyzed by 1H and 13C NMR. Tests for antiplatelet aggregation activities of all of the four compounds were conducted. RESULTS: X-ray diffraction studies show that (L1) and (L2) exist in the enol-imine tautomeric form with a strong intramolecular hydrogen bond. NMR studies show that both ligands are found as enol-imine tautomers in CDCl3 solution. In the solid state, the geometry around the copper(II) ion in both (C1) and (C2) is square planar. EPR spectra suggest that the geometry of the complexes is similar to that observed in the solid state by X-ray crystallography. Compound (C2) exhibited the strongest antiplatelet aggregation activity. CONCLUSION: Schiff base copper(II) complexes, which are attracting increasing interest, could represent a new approach to treat thrombosis by blocking the activity of PolyP with a potential anticoagulant activity and, most importantly, demonstrating no adverse bleeding events.
Subject(s)
Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Copper/chemistry , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Adult , Humans , Male , Middle Aged , Schiff Bases/chemistry , Young AdultABSTRACT
Renal cell carcinoma (RCC), also called kidney cancer or renal adenocarcinoma, is highly resistant to current treatments. It has been previously reported that a Kunitz-type inhibitor domain-containing protein, isolated from the salivary glands of the Amblyomma cajennense tick, triggers apoptosis in murine renal adenocarcinoma cells (Renca) by inhibiting the proteasome and endoplasmic reticulum stress. Of note, Amblyomin-X is the corresponding recombinant protein identified in the cDNA library from A. cajennense salivary glands. Herein, using orthotopic kidney tumors in mice, we demonstrate that Amblyomin-X is able to drastically reduce the incidence of lung metastases by inducing cell cycle arrest and apoptosis. The in vitro assays show that Amblyomin-X is capable of reducing the proliferation rate of Renca cells, promoting cell cycle arrest, and down-regulating the expression of crucial proteins (cyclin D1, Ki67 and Pgp) involved in the aggressiveness and resistance of RCC. Regarding non-tumor cells (NIH3T3), Amblyomin-X produced minor effects in the cyclin D1 levels. Interestingly, observing the image assays, the fluorescence-labelled Amblyomin-X was indeed detected in the tumor stroma whereas in healthy animals it was rapidly metabolized and excreted. Taken the findings together, Amblyomin-X can be considered as a potential anti-RCC drug candidate.
Subject(s)
Apoptosis/drug effects , Carcinoma, Renal Cell/drug therapy , Kidney Neoplasms/drug therapy , Proteasome Inhibitors/pharmacology , Salivary Proteins and Peptides/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Arthropod Proteins , Carcinoma, Renal Cell/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cyclin D1/metabolism , Down-Regulation , Endoplasmic Reticulum Stress/drug effects , Humans , Ki-67 Antigen/metabolism , Kidney/pathology , Kidney Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , NIH 3T3 Cells , Proteasome Endopeptidase Complex/drug effects , Proteasome Inhibitors/therapeutic use , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Salivary Proteins and Peptides/therapeutic use , Toxicity Tests , Xenograft Model Antitumor AssaysABSTRACT
La lipoproteína (a), Lp(a), es un factor genético de mayor riesgo para la aterosclerosis que otros factores de riesgo. La diferencia esencial entre esta lipoproteina y las lipoproteínas de baja densidad es la presencia de una apolipoproteína, la apo(a), estructuralmente parecida al plasminógeno. Esta similitud estructural le confiere la capacidad de unirse con la fibrina y a las proteínas de las membranas celulares. La Lp(a) puede interferir con la fibrinólisis, favorecer los depósitos de líquidos y estimular el crecimiento de células musculares lisas. Se admite que el efecto aterotrombogénico de la Lp(a) depende de su concentración plasmática elevada. Sin embargo, existe un aspecto cualitativo ligado a las isoformas de la apo(a) y a su importante homología estructural con el plasminógeno. En efecto, la existencia de una relación inversa entre el tamaño de las isoformas de la apo(a) y el efecto antifibrinolítico de la Lp(a), ha sido recientemente reportado. Así las isoformas más pequeñas tendrían una afinidad más elevada por la fibrina y como consecuencia, el efecto antifibrinolítico más pronunciado. Esta heterogeneidad funcional estaría ligada al polimorfismo estructural de la apo(a). En total, 34 alelos diferentes codifican a otro tanto de isoformas de apo(a), variando el tamaño entre 300 y 800 kD. Los sujetos heterocigotos consituyen el 94 por ciento de la población general y poseen en el plasma una isoforma heredada de cada progenitor, en este caso, el riesgo aterogénico resultante del efecto antifibrinolítico de la Lp(a) estaría en relación con la concentración de cada isoforma y de su afinidad relativa por la fibrina con respecto al plasminógeno(AU)
Subject(s)
In Vitro Techniques , Lipoprotein(a)/blood , Thrombosis/etiology , Atherosclerosis/etiologyABSTRACT
La lipoproteína (a), Lp(a), es un factor genético de mayor riesgo para la aterosclerosis que otros factores de riesgo. La diferencia esencial entre esta lipoproteina y las lipoproteínas de baja densidad es la presencia de una apolipoproteína, la apo(a), estructuralmente parecida al plasminógeno. Esta similitud estructural le confiere la capacidad de unirse con la fibrina y a las proteínas de las membranas celulares. La Lp(a) puede interferir con la fibrinólisis, favorecer los depósitos de líquidos y estimular el crecimiento de células musculares lisas. Se admite que el efecto aterotrombogénico de la Lp(a) depende de su concentración plasmática elevada. Sin embargo, existe un aspecto cualitativo ligado a las isoformas de la apo(a) y a su importante homología estructural con el plasminógeno. En efecto, la existencia de una relación inversa entre el tamaño de las isoformas de la apo(a) y el efecto antifibrinolítico de la Lp(a), ha sido recientemente reportado. Así las isoformas más pequeñas tendrían una afinidad más elevada por la fibrina y como consecuencia, el efecto antifibrinolítico más pronunciado. Esta heterogeneidad funcional estaría ligada al polimorfismo estructural de la apo(a). En total, 34 alelos diferentes codifican a otro tanto de isoformas de apo(a), variando el tamaño entre 300 y 800 kD. Los sujetos heterocigotos consituyen el 94 por ciento de la población general y poseen en el plasma una isoforma heredada de cada progenitor, en este caso, el riesgo aterogénico resultante del efecto antifibrinolítico de la Lp(a) estaría en relación con la concentración de cada isoforma y de su afinidad relativa por la fibrina con respecto al plasminógeno