ABSTRACT
Quinine, a quinoline derivative, is an ancient antipyretic drug with antimalarial properties that has been phased out by more effective synthetic candidates. In previous studies we discovered that hydroxychloroquine (HCQ), a synthetic antimalarial with structural similarities to quinine, reduced the binding of antiphospholipid (aPL) immune complexes to phospholipid bilayers. We performed ellipsometry and atomic force microscopy (AFM) studies to measure the effect of quinine on dissociation of anti-ß2-glycoprotein I (anti-ß2GPI) immune complexes. We found that quinine desorbed pre-formed ß2GPI-aPL immunoglobulin (Ig)G complexes from phospholipid bilayers at significantly lower molar concentrations than HCQ. Quinine also inhibited the formation of immune complexes with a higher efficacy than HCQ at equivalent drug concentrations of 0.2 mg/ml (0.192 ± 0.025 µg/cm(2) for quinine vs. 0.352 ± 0.014 µg/cm(2) for HCQ, p < 0.001). Furthermore, AFM imaging experiments revealed that addition of quinine disintegrated immune complexes bound to planar phospholipid layers. The desorptive and inhibitory effects of the old drug, quinine, toward ß2GPI-aPL IgG complexes and ß2GPI were significantly more pronounced compared to the synthetic antimalarial, HCQ. The results suggest that the quinoline core of the molecule is a critical domain for this activity and that side chains may further modulate this effect. The results also indicate that there may yet be room for considering new activities of very old drugs in devising clinical trials on potential non-anticoagulant treatments for antiphospholipid syndrome (APS).
Subject(s)
Antigen-Antibody Complex/drug effects , Antimalarials/pharmacology , Quinine/pharmacology , Antibodies, Antiphospholipid/immunology , Antigen-Antibody Complex/chemistry , Antimalarials/chemistry , Humans , Hydroxychloroquine/chemistry , Hydroxychloroquine/pharmacology , Immunoglobulin G/immunology , Immunologic Techniques , Microscopy, Atomic Force , Molecular Structure , Phospholipids/immunology , Quinine/chemistry , beta 2-Glycoprotein I/immunologyABSTRACT
The valve sinuses of the deep venous system are frequent sites of venous thrombus initiation. We previously reported that, in comparison with the non-valvular lumenal endothelium, the valve sinus endothelium had decreased expression of von Willebrand factor (vWF) and increased expression of endothelial protein C receptor (EPCR) and thrombomodulin (TM), suggesting alteration in the procoagulant/anticoagulant balance. We hypothesized that increased stasis in the deeper recesses of the venous valves would be associated with a gradient of increased thromboresistance. Expression of EPCR, TM, and vWF was analyzed via quantitative confocal immunofluorescence in residual saphenous veins collected following coronary artery bypass procedures. In agreement with our hypothesis, endothelial expression of vWF in the valve sinus decreased from the uppermost to the deepest region of the valve sinus. In contrast to our hypothesis, EPCR expression decreased from the uppermost to the deepest region of the valve sinus (p < 0.001) and TM expression remained unchanged throughout the valve sinus. Comparison of the non-valvular lumenal endothelium with the valve sinus endothelium demonstrated significantly decreased vWF expression (p < 0.001) in the valvular sinus consistent with our previous report; however, we did not observe statistically significant differences in EPCR or TM expression in this comparison. In addition, remarkable inter-individual variation in expression of these three proteins was also observed. These findings suggest that the genesis of these observations is more complex than predicted by our initial hypothesis, likely due, at least in part, to the complex rheology of the valvular sinus microenvironment.
Subject(s)
Antigens, CD/biosynthesis , Endothelium, Vascular/metabolism , Receptors, Cell Surface/biosynthesis , Thrombomodulin/biosynthesis , Venous Valves/metabolism , von Willebrand Factor/biosynthesis , Endothelial Protein C Receptor , Gene Expression , Genetic Variation , Humans , Saphenous Vein/metabolism , Thrombomodulin/metabolism , von Willebrand Factor/metabolismABSTRACT
Annexin A5 (AnxA5) binds to phospholipid bilayers, forming two-dimensional crystals that block the phospholipids from availability for coagulation enzyme reactions. Antiphospholipid (aPL) antibodies cause gaps in the ordered crystallization of AnxA5 which expose phospholipids and thereby accelerate blood coagulation reactions. The aPL antibody-mediated disruption of AnxA5 crystallization has been confirmed on artificial phospholipid bilayers and on cell membranes including endothelial cells, placental trophoblasts and platelets. Recently, we reported that hydroxychloroquine, a synthetic antimalarial drug, can reverse this antibody-mediated process through two mechanisms: (1) by inhibiting the formation of aPL IgG-beta2glycoprotein I complexes; and (2) by promoting the formation of a second layer of AnxA5 crystal 'patches' over areas where the immune complexes had disrupted AnxA5 crystallization. In another translational application, we have developed a mechanistic assay that reports resistance to AnxA5 anticoagulant activity in plasmas of patients with aPL antibodies. AnxA5 resistance may identify a subset of aPL syndrome patients for whom this is a mechanism for pregnancy losses and thrombosis. The elucidation of aPL-mediated mechanisms for thrombosis and pregnancy complications may open new paths towards addressing this disorder with targeted treatments and mechanistic assays.
Subject(s)
Annexin A5/immunology , Antibodies, Antiphospholipid/immunology , Antiphospholipid Syndrome/complications , Abortion, Habitual/etiology , Abortion, Habitual/immunology , Animals , Annexin A5/metabolism , Antiphospholipid Syndrome/immunology , Blood Coagulation/immunology , Female , Humans , Lipid Bilayers/metabolism , Phospholipids/metabolism , Pregnancy , Pregnancy Complications/etiology , Pregnancy Complications/immunology , Thrombosis/etiology , Thrombosis/immunology , beta 2-Glycoprotein I/immunologyABSTRACT
The phospholipid binding protein, annexin A5 (AnxA5), has potent anticoagulant properties that result from its forming 2-dimensional crystals over phospholipids, blocking the availability of the phospholipids for critical coagulation enzyme reactions. This article reviews the evidence that antiphospholipid antibodies can disrupt this anticoagulant shield and unmask thrombogenic anionic phospholipids, which may thereby contribute to thrombosis in patients with the antiphospholipid syndrome (APS). This mechanism for thrombosis in APS can be monitored with coagulation assays for resistance to anticoagulant activity of AnxA5.
Subject(s)
Annexin A5/physiology , Antibodies, Antiphospholipid/physiology , Antiphospholipid Syndrome/complications , Binding Sites, Antibody , Phospholipids/physiology , Thrombosis/etiology , Antiphospholipid Syndrome/pathology , Fibrinolysis/physiology , HumansABSTRACT
Acute coronary syndromes are generally precipitated by rupture of lipid-laden, relatively acellular, vulnerable atherosclerotic plaques with thin fibrous caps. We investigated whether a high-fat diet alters insulin sensitivity and whether insulin sensitizers (troglitazone and rosiglitazone) alter the composition of otherwise lipidladen atherosclerotic plaques in mice deficient in apolipoprotein E (ApoE). ApoE-knockout mice were fed a high-fat (n=30) or standard chow (n=10) diet for two weeks. Thereafter, those fed the high-fat diet were treated with troglitazone (n=10), rosiglitazone (n=10) or no drug (n=10) for 16 weeks beginning at 8 weeks of age. Carbohydrate metabolism was assessed with intraperitoneal glucose tolerance tests and insulin tolerance tests. Plaque composition was characterised with confocal laser scanning microscopy. The high-fat diet induced insulin resistance in the absence of weight gain. Compared with control animals on the high-fat diet, animals given troglitazone (400 mg/kg/day) or rosiglitazone (4 mg/kg/day) had significantly less area under the curve (AUC) for insulin ( p<0.05) and glucose disposal ( p<0.05). Despite significant increases in insulin sensitivity with drug treatment, no change in HDL-cholesterol and triglyceride levels, nor reduction in atheroma size or lipid content was noted. Thus, improvement in insulin resistance induced by a high-fat diet in this animal model of vasculopathy did not alter plaque composition.
Subject(s)
Apolipoproteins E/blood , Apolipoproteins E/deficiency , Arteriosclerosis/genetics , Arteriosclerosis/physiopathology , Hypoglycemic Agents/pharmacology , Lipids/blood , Animals , Arteriosclerosis/pathology , Carotid Stenosis/pathology , Carotid Stenosis/physiopathology , Diet, Atherogenic , Disease Susceptibility , Insulin Resistance , Mice , Mice, Knockout , Microscopy, ConfocalABSTRACT
Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM), to studies of cell signaling by environmental agents in lung disease. Using both cells in culture and lung tissue, we show that these technologies are powerful tools for understanding signal transduction cascades elicited by toxic agents, such as oxidants and asbestosfibers, and their relationship to the development of cell injury and proliferation, responses leading to lung disease and/or repair.
Subject(s)
Air Pollutants/toxicity , Lung Diseases/etiology , Lung Diseases/physiopathology , Microscopy, Confocal/methods , Animals , Biotechnology , Cell Communication , Flow Cytometry/methods , Lasers , Lung Diseases/pathology , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/metabolism , Polymerase Chain Reaction , Signal Transduction , TransfectionABSTRACT
The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. Numerous biochemical techniques have been described for its detection in cells or tissues. Although these approaches are quantitative, they do not provide insights into whether the lesion occurs in mitochrondrial versus genomic DNA. In addition, biochemical techniques are not amenable to the evaluation of individual cells or archival tissues. Antibodies have been raised against 8-oxoG, which may circumvent some of these issues. In this review, we described the use of in situ imaging techniques to detect oxidative DNA damage including the comet assay. We will review our previous work that describes the utility of an antibody fragment (Fab) engineered to recognize 8-oxoG in DNA. Furthermore, we will discuss the analysis of DNA repair enzymes in the assessment of oxidative DNA damage. Finally, advantages and potential concerns associated with immunodetection of 8-oxoG are discussed.
Subject(s)
DNA Adducts/analysis , DNA Repair , Guanine/analogs & derivatives , Guanine/analysis , Animals , HumansABSTRACT
The extracellular signal-regulated kinase (ERK) pathway is induced by cytokines and oxidative stress. In this study we examined the patterns of localization of phosphorylated ERK proteins in relationship to subsequent phenotypic responses by the mitogenic agent epidermal growth factor (EGF) (5 ng/ ml); hydrogen peroxide (H(2)O(2)) (100 to 300 microM), an inducer of apoptosis; and crocidolite asbestos (5 microg/cm(2) dish) in a nontransformed murine alveolar type II epithelial cell line (C10). Laser scanning cytometry and flow cytometry were used to determine: (1) whether expression of phosphorylated ERKs was cell cycle-related; and (2) whether cell-cycle alterations by agents could be modified after addition of the mitogen-activated protein kinase/ERK kinase (MEK) 1 inhibitor PD98059. In contrast to other stimuli which induced transient increases in phosphorylated ERKs, asbestos caused fiber-associated localization of phosphorylated ERKs that were elevated from 1 to 24 h (P < or = 0.05), and striking apoptosis followed by increased numbers of cells in the S phase at 72 h. In both control and asbestos-exposed cells, the percentage of phosphorylated ERK-positive cells was greatest in cells in the G(2)/M and S phases of the cell cycle. All stimuli caused increased proportions of cells in G(2)/M at 24 h that were inhibited by PD98059 (30 microM). Increases in G(2)/M cells by H(2)O(2) and asbestos also were decreased at 48 h by the MEK1 inhibitor. In addition, PD98059 abrogated elevations in S-phase cells by EGF and H(2)O(2) at 24 h and by asbestos at 72 h. Our results suggest that ERKs mediate cell-cycle alterations during the development of epithelial cell apoptosis or proliferation by diverse ERK stimuli.
Subject(s)
Asbestos/pharmacology , Carcinogens/pharmacology , Epidermal Growth Factor/pharmacology , Hydrogen Peroxide/pharmacology , Lung/cytology , MAP Kinase Signaling System/drug effects , Oxidants/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Division/drug effects , Cell Division/physiology , Enzyme Inhibitors/pharmacology , Epithelial Cells/chemistry , Epithelial Cells/cytology , Epithelial Cells/enzymology , Extracellular Space/enzymology , Flavonoids/pharmacology , Lung/metabolism , MAP Kinase Signaling System/physiology , Mice , Microscopy, Confocal , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Proliferating Cell Nuclear Antigen/analysisABSTRACT
Aspirin and abciximab independently decrease the incidence of cardiac events. To identify potential interactions, antiplatelet effects of abciximab were characterized in blood from healthy subjects given aspirin. Platelet activation was determined in whole blood with and without abciximab (2 microg/ml) added in vitro. Flow cytometry was used to quantify fibrinogen binding (glycoprotein IIb-IIIa activation). Binding of fluorochrome-labeled and 125I-labeled abciximab was determined before and after exposure to aspirin. In blood from subjects given aspirin for 5 days, abciximab-induced inhibition of the capacity to bind fibrinogen in response to 1 microM ADP was greater when the daily dose had been 325 mg compared with 81 mg (% inhibition: no aspirin 53 +/- 6; 81 mg daily 62 +/- 5; 325 mg daily 69 +/- 6). The effect of 5 daily doses of aspirin was greater than that of one. Larger single doses elicited larger effects (% inhibition 2 h after 325 mg 59 +/- 6; 2 h after 650 mg 78 +/- 5). Neither salicylsalicylic acid nor naproxen sodium potentiated the effect of abciximab. Exposure of platelets to 14C-acetylsalicylic acid led to acetylation of glycoprotein IIb and IIIa. Binding of 125I-abciximab to platelets was increased after 30 and 60 min. Acetylation of glycoprotein IIb-IIIa by aspirin augments inhibitory effects of abciximab in a dose- and time-dependent manner by increasing binding of abciximab to platelets.
Subject(s)
Antibodies, Monoclonal/pharmacology , Aspirin/pharmacology , Immunoglobulin Fab Fragments/pharmacology , Platelet Activation/drug effects , Abciximab , Acetylation , Antibodies, Monoclonal/pharmacokinetics , Aspirin/administration & dosage , Aspirin/pharmacokinetics , Carbon Radioisotopes , Dose-Response Relationship, Drug , Drug Synergism , Fibrinogen/metabolism , Humans , Iodine Radioisotopes , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Aggregation Inhibitors/pharmacology , Platelet Function Tests , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein Binding/drug effects , Time FactorsABSTRACT
Recent and new results which support a drug-DNA covalent bonding mechanism for cell toxicity of the clinical antitumor drugs, daunorubicin, doxorubicin, and epidoxorubicin, are summarized. The mechanism involves the iron complex of the drugs inducing oxidative stress to yield formaldehyde, which then mediates covalent attachment to G-bases of DNA. At NGC sites the combination of covalent and non-covalent drug interactions serve to virtually crosslink the DNA. Structural data for virtual crosslinks are compared as a function of drug structure. Elucidation of the mechanism led to the synthesis and evaluation of drug formaldehyde conjugates, Daunoform, Doxoform, and Epidoxoform, as improved chemotherapeutics. Drug uptake, nuclear targeting, drug release, and cytotoxicity of the clinical drugs by sensitive and resistant breast and prostate cancer cells are contrasted with those of the corresponding formaldehyde conjugates. Conjugates are taken up better, retained longer, and are more toxic to a wide variety of tumor cells. The kinetics of drug release from Doxoform and Epidoxoform treated MCF-7/Adr cells are biexponential and correlate with the biexponential kinetics of drug release from extracellular DNA. The results of the lead conjugate, Epidoxoform, in the National Cancer Institute 60 human tumor cell screen are presented and discussed in terms of some resistance mechanisms. Epidoxoform shows increased toxicity to all panels relative to doxorubicin and epidoxorubicin, and this enhanced toxicity is especially evident with the more resistant cell lines.
Subject(s)
Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/pharmacology , Cell Nucleus/drug effects , Neoplasms/drug therapy , Neoplasms/ultrastructure , Animals , Cell Nucleus/metabolism , Drug Resistance, Neoplasm , Humans , Neoplasms/metabolism , Tumor Cells, CulturedABSTRACT
Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers. Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species (ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo). We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbers of cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmission electron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestos alone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strand breakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelial cells of the respiratory tract, thus validating in vitro observations from a number of laboratories.
Subject(s)
Asbestos, Amosite/toxicity , Bronchi/drug effects , DNA Damage , Nicotiana , Plants, Toxic , Smoke/adverse effects , Animals , Apoptosis/drug effects , Bronchi/cytology , Cocarcinogenesis , Drug Synergism , Epithelial Cells/chemistry , Epithelial Cells/drug effects , In Situ Nick-End Labeling , Male , Necrosis , Rats , Rats, Sprague-Dawley , Reactive Oxygen SpeciesABSTRACT
Selected ion flow tube-chemical ionization mass spectrometry was used to measure formaldehyde levels in human breast cancer cells in comparison with levels in cells treated with the antitumor drugs doxorubicin (DOX) and daunorubicin (DAU) and the daunorubicin-formaldehyde conjugate Daunoform (DAUF). The measurement was performed on cell lysates and showed only background levels of formaldehyde in untreated cells and drug-treated resistant cells (MCF-7/Adr cells) but levels above background in DOX- and DAU-treated sensitive cells (MCF-7 cells). The level of formaldehyde above background was a function of drug concentration (0.5-50 microM), treatment time (3-24 h), cell density (0.3 x 10(6) to 7 x 10(6) cells/mL), and cell viability (0-100%). Higher levels of formaldehyde were observed in lysates of MCF-7 cells treated at higher drug levels, unless the treatment resulted in low cell viability. Elevated levels were directly related to cell density and were observed even with 0.5 microM drug. A lower limit for excess formaldehyde in MCF-7 cells treated with 0.5 microM DAU for 24 h is 0.3 mM. Control experiments showed that formaldehyde was not produced after cell lysis. Lysates of sensitive and resistant cells treated with 0.5 micromolar equiv of the formaldehyde conjugate (DAUF) for 3 h showed only background levels of formaldehyde. The results support a mechanism for drug cytotoxicity which involves drug induction of metabolic processes leading to formaldehyde production followed by drug utilization of formaldehyde to virtually cross-link DNA.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Doxorubicin/pharmacology , Formaldehyde/metabolism , Tumor Cells, Cultured/drug effects , Animals , Cross-Linking Reagents , Daunorubicin/pharmacology , Female , Humans , Mass SpectrometryABSTRACT
The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.
Subject(s)
Cell Nucleus/chemistry , DNA/chemistry , Guanine/analogs & derivatives , Mitochondria/chemistry , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Biomarkers/analysis , Cells, Cultured , Combinatorial Chemistry Techniques , Fluorescent Antibody Technique , Guanine/analysis , Guanine/immunology , Hemocyanins/immunology , Humans , Hydrogen Peroxide/pharmacology , Mice , Microscopy, Confocal , Oligodeoxyribonucleotides/immunology , Oxidative Stress , Rats , Recombinant Proteins/geneticsABSTRACT
To quantitatively characterize contributions of major constituents to the composition of a given atherosclerotic plaque, we have developed an approach employing immunohistochemistry, confocal scanning laser microscopy, and computer-assisted image analysis. The method developed permits identification of plaques that are particularly vulnerable to rupture and elucidation of the nature of the composition of a given plaque, as well as the extent of luminal encroachment. Thus, it should be useful in experimental animals and ultimately in patients in delineating compositional changes in response to potentially deleterious genetic and environmentally induced factors and to potentially therapeutic interventions designed to diminish plaque vulnerability.
Subject(s)
Arteriosclerosis/pathology , Coronary Vessels/pathology , Image Processing, Computer-Assisted/methods , Immunohistochemistry/methods , Microscopy, Confocal/methods , Animals , Arteriosclerosis/metabolism , Collagen/metabolism , Coronary Vessels/metabolism , Fluorescent Dyes , Lipid Metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant StrainsABSTRACT
Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.
Subject(s)
Asbestos, Serpentine/adverse effects , Asbestosis/enzymology , Asbestosis/pathology , Lung/enzymology , Lung/pathology , Mitogen-Activated Protein Kinases/metabolism , Administration, Inhalation , Animals , Asbestos, Serpentine/administration & dosage , Asbestosis/etiology , Asbestosis/metabolism , Bromodeoxyuridine/metabolism , Cell Division , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Phosphorylation , Tissue DistributionABSTRACT
OBJECTIVE: To determine whether glycoprotein (GP) IIb-IIIa inhibitors can paradoxically augment activation of platelets, activation of GP IIb-IIIa, alpha-granule degranulation, and lysosome release were induced after exposure of platelets to GP IIb-IIIa inhibitors. METHODS: ADP-induced platelet activation was assessed after exposure of platelets to Abciximab, or to a non-peptide ligand, the free acid of Orbofiban (Orbofiban(a)). Activation of GP IIb-IIIa was detected based on binding of fluorochrome labeled fibrinogen or a labeled monoclonal antibody, PAC-1. alpha-Granule degranulation was detected based on surface expression of P-selectin and lysosome release was detected based on surface expression of CD63. RESULTS: Despite significant inter-individual variability in inhibition of fibrinogen binding in response to each of the GP IIb-IIIa inhibitors used, a concentration dependent decrease in fibrinogen binding was seen with each agent in samples from each subject. Binding of PAC-1 was inhibited in a parallel manner. Abciximab increased ADP-induced P-selectin expression. Orbofiban(a) did not alter ADP-induced P-selectin expression. Neither agent altered ADP-induced CD63 expression. When platelets were exposed to Abciximab and Orbofiban(a), both Abciximab and Orbofiban(a) were found in the alpha-granules (by confocal microscopy), consistent with potentiation of agonist-induced release of alpha-granular products associated with uptake of proteins. CONCLUSIONS: Specific types of GP IIb-IIIa inhibitors can paradoxically augment agonist-induced release of alpha-granules despite inhibiting agonist-induced fibrinogen binding.
Subject(s)
Antibodies, Monoclonal/pharmacology , Blood Platelets/drug effects , Fibrinogen/metabolism , Immunoglobulin Fab Fragments/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Pyrrolidines/pharmacology , Abciximab , Alanine/metabolism , Alanine/pharmacology , Antibodies, Monoclonal/metabolism , Antigens, CD/analysis , Blood Platelets/immunology , Blood Platelets/metabolism , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Immunoglobulin Fab Fragments/metabolism , In Vitro Techniques , Microscopy, Confocal , P-Selectin/analysis , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/metabolism , Platelet Membrane Glycoproteins/analysis , Protein Binding/drug effects , Pyrrolidines/metabolism , Tetraspanin 30ABSTRACT
Epidoxorubicin and daunorubicin are proposed to be cytotoxic to tumor cells by catalyzing production of formaldehyde through redox cycling and using the formaldehyde for covalent attachment to DNA at G bases. The crystal structure of epidoxorubicin covalently bound to a d(CGCGCG) oligomer was determined to 1.6 A resolution. The structure reveals slightly distorted B-form DNA bearing two molecules of epidoxorubicin symmetrically intercalated at the termini, with each covalently attached from its N3' to N2 of a G base via a CH2 group from the formaldehyde. The structure is analogous to daunorubicin covalently bound to d(CGCGCG) determined previously, except for additional hydrogen bonding from the epimeric O4' to O2 of a C base. The role of drug-DNA covalent bonding in cells was investigated with synthetic epidoxorubicin-formaldehyde conjugate (Epidoxoform) and synthetic daunorubicin-formaldehyde conjugate (Daunoform). Uptake and location of drug fluorophore in doxorubicin-resistant human breast-cancer cells (MCF-7/ADR cells) was observed by fluorescence microscopy and flow cytometry. The fluorophore of Daunoform appeared more rapidly in cells and was released more rapidly from cells than the fluorophore of Epidoxoform over a 3 h exposure period. The fluorophore appeared predominantly in the nucleus of cells treated with both conjugates. The difference in uptake is explained in terms of the slower rate of hydrolysis of Epidoxoform to the species reactive with DNA and a proposed slower release from DNA based upon the crystal structures.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/chemistry , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA, Neoplasm/metabolism , Epirubicin/analogs & derivatives , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Breast Neoplasms/drug therapy , Cross-Linking Reagents/chemistry , Crystallography, X-Ray , Daunorubicin/analogs & derivatives , Daunorubicin/chemistry , Daunorubicin/metabolism , Daunorubicin/pharmacokinetics , Daunorubicin/pharmacology , Epirubicin/chemistry , Epirubicin/metabolism , Epirubicin/pharmacokinetics , Epirubicin/pharmacology , Humans , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Intercalating Agents/pharmacokinetics , Intercalating Agents/pharmacology , Nucleic Acid Conformation , Oligonucleotides/chemistry , Tissue Distribution , Tumor Cells, CulturedABSTRACT
BACKGROUND: Intracranial bleeding is encountered in some patients with acute myocardial infarction treated with fibrinolytic drugs, and especially in patients with occult cerebral vasculopathy. In order to determine whether pharmacologically induced plasminemia is a determinant, and whether impairment of the blood-brain barrier can serve as a marker of risk, we studied spontaneously hypertensive stroke-prone rats (SHRSP) genetically disposed to cerebral vasculopathy. METHODS: In order to simulate the induction of plasminemia in patients treated with fibrinolytic drugs for acute myocardial infarction, three intravenous injections of human plasminogen and human tissue-type plasminogen activator (t-PA) were administered to the rats at 2-h intervals (12 mg plasminogen plus 60 micrograms t-PA, 6 mg plus 30 micrograms t-PA, and 0.5 mg plus 2.5 micrograms t-PA), and serial blood samples were assayed for plasmin. One hour after the final intravenous injection, the brain was perfusion-fixed with 4% formaldehyde, and the blood-brain barrier integrity was assessed by localization of immunoglobulin G (IgG) using fluorescein-conjugated goat anti-rat IgG and confocal microscopy. RESULTS: Transient plasminemia followed each injection of plasminogen and t-PA. Intracranial extravasation of IgG was observed in nine of 11 SHRSP treated with t-PA and plasminogen. None of seven SHRSP injected with vehicle alone exhibited extravasation (chi 2 = 6.37, P = 0.01). CONCLUSIONS: The results indicate that the blood-brain barrier in SHRSP is susceptible to impairment secondary to pharmacologically induced plasminemia and is predisposed to bleeding. The preparation developed may facilitate the delineation of relative risk of injury to cerebral vessels with thrombolytic therapy.
Subject(s)
Blood-Brain Barrier , Brain/drug effects , Cerebral Hemorrhage/physiopathology , Fibrinolytic Agents/pharmacology , Hypertension/physiopathology , Animals , Brain/metabolism , Brain/physiopathology , Cerebral Hemorrhage/chemically induced , Cerebral Hemorrhage/metabolism , Extravasation of Diagnostic and Therapeutic Materials , Female , Fibrinolysin/metabolism , Hypertension/metabolism , Immunoglobulin G/metabolism , Male , Microscopy, Confocal , Plasminogen/pharmacology , Random Allocation , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Tissue Plasminogen Activator/pharmacologyABSTRACT
The anthracycline, antitumor drugs doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI) catalyze production of formaldehyde through induction of oxidative stress. The formaldehyde then mediates covalent bonding of the drugs to DNA. Synthetic formaldehyde conjugates of DOX, DAU, and EPI, denoted Doxoform (DOXF), Daunoform (DAUF), and Epidoxoform (EPIF), exhibit enhanced toxicity to anthracycline-sensitive and -resistant tumor cells. Uptake and retention of parent anthracycline antitumor drugs (DOX, DAU, and EPI) relative to those of their formaldehyde conjugates (DOXF, DAUF, and EPIF) were assessed by flow cytometry in both drug-sensitive MCF-7 cells and drug-resistant MCF-7/ADR cells. The MCF-7 cells took up more than twice as much drug as the MCF-7/ADR cells, and both cell lines took up substantially more of the formaldehyde conjugates than the parent drugs. Both MCF-7 and MCF-7/ADR cells retained fluorophore from DOXF, DAUF, and EPIF hours after drug removal, while both cell lines almost completely expelled DOX, DAU, and EPI within 1 h. Longer treatment with DOX, DAU, and EPI resulted in modest drug retention in MCF-7 cells following drug removal but poor retention of DOX, DAU, and EPI in MCF-7/ADR cells. Fluorescence microscopy showed that the formaldehyde conjugates targeted the nuclei of both sensitive and resistant cells, and remained in the nucleus hours after drug removal. Experiments in which [(3)H]Doxoform was used, synthesized from doxorubicin and [(3)H]formaldehyde, also indicated that Doxoform targeted the nucleus. Elevated levels of (3)H were observed in DNA isolated from [(3)H]Doxoform-treated MCF-7 and MCF-7/ADR cells relative to controls. The results implicate drug-DNA covalent bonding in the tumor cell toxicity mechanism of these anthracyclines.
Subject(s)
Antineoplastic Agents/pharmacology , DNA, Neoplasm/metabolism , Daunorubicin/pharmacology , Doxorubicin/pharmacology , Epirubicin/pharmacology , Formaldehyde/metabolism , Alkylation , Antineoplastic Agents/metabolism , Cell Nucleus/metabolism , DNA, Neoplasm/analysis , Daunorubicin/analogs & derivatives , Daunorubicin/metabolism , Doxorubicin/analogs & derivatives , Doxorubicin/metabolism , Epirubicin/analogs & derivatives , Epirubicin/metabolism , Humans , RNA, Neoplasm/analysis , Spectrometry, Fluorescence , Tumor Cells, CulturedABSTRACT
Recent data indicate that the clinical anthracycline anti-tumor drugs, doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI), catalyze the production of formaldehyde through induction of oxidative stress and bind the formaldehyde to form a metabolite which covalently bonds to DNA. Based upon this discovery, anthracycline-formaldehyde conjugates were synthesized and evaluated in three metastatic prostate cancer cell lines, LNCaP, PC-3, and DU-145. The doxorubicin-formaldehyde conjugate, Doxoform (DOXF), inhibits the growth of PC-3 and DU-145 cells 50- and 80-fold better, respectively, than the corresponding clinical drug, DOX. Daunorubicin- and epidoxorubicin-formaldehyde conjugates, Daunoform and Epidoxoform (DAUF and EPIF), inhibit the growth about 6 to 10-fold better than the clinical drugs, DAU and EPI. In addition, DAUF, DOXF, and EPIF are 2- to 20-fold more toxic to the doxorubicin-sensitive metastatic prostate cancer cell line, LNCaP. Fluorescence microscopy indicates that the nucleus is the major target for all six drugs. Flow cytometry together with fluorescence microscopy shows that DOXF and EPIF are taken up more rapidly and more abundantly and are retained in the nucleus longer than DOX and EPI, respectively, especially in DU-145 cells. The enhanced toxicity of the anthracycline-formaldehyde conjugates is attributed to their increased nuclear uptake and retention and suggests that DOXF, DAUF, and EPIF are prodrugs to the active metabolites of the clinical drugs DOX, DAU, and EPI.