dicumarol and Hypoxia

The overexpression of hypoxia-inducible factor-1 alpha (HIF-1α) in solid tumor compromises the potency of chemotherapy under hypoxia. The high level of HIF-1α arises from the stabilization effect of reduced nicotinamideadeninedinucleotide(phosphate) NAD(P)H: quinone oxidoreductase 1 (NQO1). It was postulated that the inhibition of NQO1 could degrade HIF-1α and sensitize hypoxic cancer cells to antineoplastic agents. In the current work, we report hypoxia-responsive polymer micelles, i.e. methoxyl poly(ethylene glycol)-co-poly(aspartate-nitroimidazole) orchestrate with a NQO1 inhibitor (dicoumarol) to sensitize the ovarian cancer cell line (SKOV3) to a model anticancer agent (sorafenib) at low oxygen conditions. Both cargos were physically encapsulated in the nanoscale micelles. The placebo micelles transiently induced the depletion of reduced nicotinamideadeninedinucleotidephosphate (NADPH) as well as glutathione and thioredoxin under hypoxia, which further inactivated NQO1 because NADPH was the cofactor of NQO1. As a consequence, the expression of HIF-1α was repressed due to the dual action of dicoumarol and polymer. The degradation of HIF-1α significantly increased the vulnerability of SKOV3 cells to sorafenib-induced apoptosis, as indicated by the enhancement of cytotoxicity, and increase of caspase 3 and cytochrome C. The current work opens new avenues of addressing hypoxia-induced drug resistance in chemotherapy.

Topics: Antineoplastic Agents; Aspartic Acid; Caspase 3; Cell Hypoxia; Cell Line, Tumor; Cytochromes c; Dicumarol; Female; Glutathione; Humans; Hypoxia; Micelles; NAD; NADP; Nitroimidazoles; Oxygen; Phosphates; Polyethylene Glycols; Polymers; Quinones; Sorafenib; Thioredoxins

Some derivatives of phenazine 5,10-dioxide are selectively toxic to hypoxic cells commonly found in solid tumors. Previous studies of the phenazine 5,10-dioxide mechanism of action indicated that a bioreduction process could be involved in its selective toxicities, maybe as result of its potential H(*)-releasing capability in hypoxia. The major unresolved aspect of the mechanism of phenazine 5,10-dioxides is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form and metabolites. We have studied the metabolism in both hypoxia and oxia of some selected 2-amino and 2-hydroxyphenazine 5,10-dioxides, 1- 5, using rat liver microsomal and cytosol fractions. Differential hypoxic/oxic metabolism was found to be correlated to a compound's cytotoxic selectivity but, in general, without metabolic differences between liver microsomal or cytosolic enzymes. Dicoumarol and ketoconazole were found to inhibit the hypoxic metabolism of the most selective phenazine 5,10-dioxide, 1, inferring a role for DT-diaphorase and cytochrome P450. The least hypoxic selective agents, 4 and 5, possess different hypoxia-metabolic profiles as compared to derivative 1, explaining the differential cytotoxic biological behavior. The nonselective derivative, 2, suffered bioreduction in both conditions and, according to the inhibition studies with dicoumarol and ketoconazole, involves both DT-diaphorase and cytochrome P450. The nontoxic derivative, 3, showed poor bioreductive behavior.

Topics: Aldehyde Oxidase; Animals; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; Cytosol; Dicumarol; Hypoxia; Ketoconazole; Male; Microsomes, Liver; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Phenazines; Rats; Rats, Sprague-Dawley; Stereoisomerism; Structure-Activity Relationship

Baicalin and its aglycone baicalein are the major flavonoid components of the root of Scutellaria baicalensis. Recent studies have shown that they can attenuate oxidative stress in various in vitro models as they possess potent antioxidant activities. This study investigated alternative protective mechanisms of baicalein in a cardiomyocyte model.. Neonatal rat cardiomyocytes pretreated with the test compound were subjected to hypoxia/reoxygenation. The extent of cellular damage was accessed by the amount of released lactate dehydrogenase. Pretreatment with baicalein up to 10 microM reduced lactate dehydrogenase release significantly (P<0.001), while pretreatment with baicalin up to 100 microM was ineffective. The cardioprotective effect of baicalein is not due to its antioxidant effect, because an adverse effect rather than a protective effect was observed when baicalein was present during hypoxia. Cotreatment with N-acetylcysteine attenuated the protective effect of baicalein and concomitantly increased intracellular reactive oxygen species level and the cytotoxic effect of baicalein, but N-acetylcysteine alone did not have such effects. In addition, cotreatment with catalase, but not superoxide dismutase or mannitol, reversed the cardioprotective effect of baicalein, suggesting the involvement of hydrogen peroxide in the cardioprotective mechanism. The NAD(P)H:quinone oxidoreductase inhibitors dicoumarol and chrysin also abolished the cardioprotective effect of baicalein. While pretreatment with baicalein did not increase antioxidant enzyme activities, it alleviated calcium accumulation in cardiomyocytes undergoing simulated ischemia.. These results highlight the important role of hydrogen peroxide produced during the auto-oxidation of baicalein in the cardioprotective effect of baicalein.

Topics: Acetylcysteine; Animals; Antioxidants; Calcium; Catalase; Cells, Cultured; Dicumarol; Enzyme Inhibitors; Flavanones; Flavonoids; Hypoxia; L-Lactate Dehydrogenase; Myocytes, Cardiac; Rats

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) is an anti-tumour prodrug which recently entered clinical trials in combination with Escherichia coli nitroreductase in a gene-directed enzyme prodrug therapy (GDEPT) context. A Phase I trial of the prodrug, however, revealed dose-limiting hepatotoxicity (transaminitis). The aim of this study was to find out whether the prodrug undergoes reductive metabolism in human liver to cytotoxic metabolites which may contribute to this clinical toxicity. CB 1954 (2.5-250 microM) was incubated with human liver preparations (2-8 mg/mL of S9, cytosolic or microsomal proteins) in the presence of NAD(P)H (1 mM). The NADH- and NADPH-dependent formation of both 2- and 4-nitroreduction products was demonstrated, with NADPH being the preferred cofactor, by HPLC and mass spectrometry. The major metabolite formed in all three human liver preparations was the 4-hydroxylamine, a potent DNA cross-linking cytotoxin. The 2-hydroxylamine and 2-amine metabolites were also detected, both of which have also been demonstrated to be highly cytotoxic. 2-Nitroreduction was far greater in S9 compared with cytosol and was not detected in microsomal preparations. Although 2- and 4-nitroreduction of CB 1954 was inhibited under hyperoxic conditions, substantial metabolism was observed under atmospheric oxygen levels. These studies demonstrate that human liver is capable of aerobic reductive bioactivation of CB 1954 to cytotoxic metabolites in vitro, possibly involving multiple enzymes, which may account for the clinical hepatotoxicity observed.

Topics: Antineoplastic Agents; Aziridines; Carbon Monoxide; Chromatography, High Pressure Liquid; Clinical Trials, Phase I as Topic; Cytosol; Cytotoxins; Dicumarol; Escherichia coli; Humans; Hydroxylamines; Hypoxia; Liver; Mass Spectrometry; Microsomes, Liver; NAD; NADP; Nitroreductases; Prodrugs

Aerobic and hypoxic cultures of EMT6 mouse mammary tumor cells were used to study the effects of dicoumarol (DIC) on the cytotoxicity of mitomycin C (MC). DIC protected aerobic cells from MC toxicity, but sensitized hypoxic cells to the cytotoxic actions of this antibiotic. Survival curves for cells treated with 1.5 microM MC +/- 100 microM DIC for different periods of time under aerobic or hypoxic conditions showed that DIC acted as a dose-modifying agent, that is, an agent which changed the slopes, but not the shapes, of the MC survival curves. Experiments that examined the effects of the DIC concentration on the modulation of MC cytotoxicity revealed significant perturbations in MC toxicity with a DIC concentration of 100 microM and increased sensitization/protection with increasing levels of DIC. DIC altered the toxicity of MC only when it was present during exposure of the cells to MC. Treatment with DIC before or after (but not during) MC did not alter the amount of cytotoxicity. Addition of DIC to cell cultures seconds before the addition of MC was as effective as addition of DIC 30 min to 2 h before MC. Taken together, these findings suggest that DIC reversibly inhibits one or more enzymes involved in the activation and inactivation of MC, and that this modulation of the enzymatic processing of MC alters the cytotoxicity of the drug.

Topics: Animals; Cell Line; Cell Survival; Dicumarol; Drug Synergism; Hypoxia; Mammary Neoplasms, Experimental; Mice; Mitomycin; Mitomycins; Tumor Cells, Cultured

Mitochondrial calcium uptake, but not binding, like microsomal calcium uptake in failing human hearts, was less than the control values for dog, rabbit, and hamster hearts. Decrease in mitochondrial calcium binding and uptake was observed in genetically myopathic hamsters (BIO 14.6) at early, moderate, and late stages of congestive heart failure. Inhibitors of mitochondrial calcium transport, Dicumarol, dinitrophenol, and sodium azide, were found to produce a rapid fall in contractility of the isolated rat heart. Inability of rat hearts to generate contractile force on perfusion with Na+- or K+-free medium was associated with an increase in mitochondrial calcium uptake. A dramatic increase in mitochondrial calcium uptake was observed on perfusing rat hearts with control medium after CA++-free medium. No change in mitochondrial calcium uptake was noted in acute ischemic dog myocardium or hypoxic rat heart in which contractile force was severely depressed. Both mitochondrial calcium transport and contractility were decreased on perfusing rat hearts with substrate-free medium; however, the change in calcium uptake was secondary to the fall in contractile force. Decrease in pH, ATP:ADP ratio, ATP6AMP ratio, and K+:Na+ ratio were found to reduce the dog heart mitochondrial calcium uptake. It is likely that various factors such as pH, ATP:ADP ration, ATP:AMP ratio, and K+ :Na+ ration, in addition to damage in mitochondrial structure, play an important role in inhibiting mitochondrial calcium transport in failing hearts. The results also suggest that alterations in mitochondrial calcium transport are dependent upon the degree and type of heart failure.

Topics: Adenine Nucleotides; Animals; Azides; Binding Sites; Biological Transport; Calcium; Cricetinae; Dicumarol; Dinitrophenols; Dogs; Heart Failure; Humans; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Metals, Alkali; Mitochondria, Muscle; Myocardial Contraction; Myocardium; Papillary Muscles; Rabbits; Sarcoplasmic Reticulum

Topics: Animals; Carbohydrates; Carbon Isotopes; Diaphragm; Dicumarol; Extracellular Space; Female; Hypoxia; In Vitro Techniques; Insulin; Male; Muscles; Organ Size; Oxygen Consumption; Photometry; Rats; Sorbitol; Time Factors; Vitamin K; Water; Xylose

Topics: Animals; Carbohydrates; Carbon Isotopes; Diaphragm; Dicumarol; Extracellular Space; Female; Fructose; Galactose; Glucose; Hexoses; Hypoxia; In Vitro Techniques; Insulin; Male; Monosaccharides; Muscles; Pentoses; Rats; Time Factors; Xylose

Topics: Arteriovenous Fistula; Blood Coagulation Disorders; Dicumarol; Female; Hemodynamics; Humans; Hypoxia; Middle Aged; Pulmonary Circulation; Pulmonary Embolism

Topics: Adenine; Adenine Nucleotides; Adenosine Triphosphate; Aminohydrolases; Animals; Azaguanine; Dicumarol; Enzyme Inhibitors; Hypoxanthines; Hypoxia; Metabolism; Myocardium; Nucleosides; Nucleotides; Oxidative Phosphorylation; Perfusion; Pharmacology; Rabbits; Research