RESUMO
Selenium exists in a number of forms with differing valence states, some of which have shown antitumor activity. We studied the tumoricidal activity of four currently available selenium forms against a human leukemia cell line and exploited the differences among them to investigate the mechanism of antitumor action. Only selenocystine and sodium selenite showed antitumor activity, and these were also the only compounds which demonstrated significant redox chemistry, including depletion of cellular glutathione, stimulation of glutathione reductase, and stimulation of oxygen consumption. The interaction of these two compounds with glutathione suggests an intriguing potential role for them in cancer therapy.
Assuntos
Glutationa/metabolismo , Compostos Organosselênicos , Selênio/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cistina/análogos & derivados , Cistina/toxicidade , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Humanos , Leucemia Experimental/patologia , Leucemia Experimental/fisiopatologia , NADP , Consumo de Oxigênio/efeitos dos fármacos , Ácido SeleniosoRESUMO
Oxygen derived free radicals and peroxides result from many antitumor treatments, including radiation and anthracyclines. Doxorubicin cardiotoxicity is thought to result from free radical induced lipid peroxidation. The heart has less active detoxification enzymes than does the liver and depends on selenium dependent glutathione peroxidase (GSH-PX) for this function. We did a sequential prospective trial in patients with totally controlled parenteral diets to examine the activity of red blood cell GSH-PX in patients with and without malignant disease. Decreased GSH-PX activity was found in 54% of the patients on parenteral nutrition and was more common in the older of these patients and in those with the greatest weight loss. In the absence of selenium supplementation, the RBC GSH-PX activity declines steadily, but with supplementation this was prevented or reversed. Because selenium deficiency can manifest as a cardiomyopathy, we measured the enzyme activity in the hearts of five patients who had died. The cardiac enzyme activity correlated strongly with the RBC levels. Significantly decreased GSH-PX has been shown in animals to be associated with changes in other enzymes critical both to activation and detoxification of carcinogens as well as antitumor drugs. Abnormality of selenium status might be a previously unsuspected contributor to interpatient variation in drug effects.
Assuntos
Eritrócitos/enzimologia , Glutationa Peroxidase/análise , Miocárdio/enzimologia , Nutrição Parenteral Total , Adulto , Peso Corporal , Feminino , Glutationa Peroxidase/sangue , Glutationa Transferase/análise , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Neoplasias/enzimologia , Distribuição Aleatória , Selenometionina/farmacologiaRESUMO
Most tumors are resistant to therapy by thymidylate synthase (TS) inhibitors due to their high levels of TS. Instead of inhibiting TS, we hypothesized that it was possible to use this enzyme to activate suicide prodrugs (deoxyuridine analogues) to more toxic species (thymidine analogues). Tumors with high levels of TS could be particularly sensitive to deoxyuridine analogues because they would be more efficient in producing the toxic methylated species. Furthermore, the accumulation of methylated species within tumors could be visualized externally if a tracer dose of the deoxyuridine analogue was tagged with an isotope, preferably a positron emitter, such as 18F. Higher accumulation of isotope indicates higher activity of TS and lower sensitivity of the tumor to TS inhibitors, but perhaps more sensitivity to therapy with deoxyuridine analogues as suicide prodrugs. 2'-F-ara-deoxyuridine (FAU) was used as a prototype to demonstrate these concepts experimentally. FAU readily entered cells and was phosphorylated, methylated, and subsequently incorporated into cellular DNA. Among different cell lines, FAU produced varying degrees of growth inhibition. Greater DNA incorporation (e.g., for CEM and U-937 cells) was reflected as increased toxicity. FAU produced less DNA incorporation in Raji or L1210 cells, and growth rate was minimally decreased. As the first demonstration that cells with high levels of TS activity can be more vulnerable to therapy than cells with low TS activity, this preliminary work suggests a new therapeutic approach for common human tumors that were previously resistant. Furthermore, it appears that the TS activity of tumors could be noninvasively imaged in situ by tracer doses of [18F]FAU and that this phenotypic information could guide patient therapy.
Assuntos
Floxuridina/metabolismo , Neoplasias/metabolismo , Pró-Fármacos/metabolismo , Timidilato Sintase/metabolismo , Animais , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/metabolismo , Arabinofuranosiluracila/farmacologia , Divisão Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/metabolismo , Radioisótopos de Flúor , Humanos , Metilação , Camundongos , Neoplasias/patologia , Fosforilação , Pró-Fármacos/farmacocinética , Células Tumorais CultivadasRESUMO
Quantitative ligand binding studies have been used to characterize binding sites for N-allylnormetazocine ((+)SKF10,047) (SKF), 1-(1-phenylcyclohexyl) piperidine (PCP), N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and haloperidol in membranes from the brain of rat and guinea pig under conditions which permitted simultaneous analysis of the binding of both PCP and SKF. Using four labelled ligands (SKF, TCP, PCP and haloperidol), each displaced by the corresponding four unlabelled ligands, four classes of binding sites were observed in membranes from the brain of the rat, corresponding to sigma (sigma), two classes of PCP sites (PCP1, PCP2) and dopamine (D2) sites. The sigma site was suppressed by 50 nM haloperidol, while the PCP1 and PCP2 sites were not. These results were confirmed by studies employing a self- and cross-displacement design and dose-response surfaces for SKF and TCP, with and without blockade by haloperidol of the sigma site. Using mathematical modelling, employing the program LIGAND, it was possible to reject simpler models involving a common "PCP/sigma" site or a model involving only two classes of sites (sigma and PCP). Similar methods were used to identify two classes of sigma binding sites and two classes of PCP binding sites, in membranes prepared from the brain of the guinea pig. The relative potencies of 18 ligands for displacement of (+)[3H]SKF10,047 and [3H]TCP were compared: there were significant qualitative and quantitative differences in the "sigma" binding sites in the brain of rat and guinea pig, while the PCP binding sites were very similar in the two species.
Assuntos
Encéfalo/metabolismo , Fenazocina/análogos & derivados , Fenciclidina/metabolismo , Receptores Opioides/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Cobaias , Masculino , Fenazocina/metabolismo , Fenciclidina/análogos & derivados , Psicotrópicos/farmacologia , Ensaio Radioligante , Ratos , Ratos Endogâmicos , Receptores Opioides/efeitos dos fármacos , Receptores sigmaRESUMO
Rat brain synaptosomal membranes that are depleted of endogenous excitatory amino acids cannot bind [(+)-5-methyl-10, 11-dihydro-5H-dibenzo(a,d]cyclohept-5,10-imine maleate] ([3H]MK-801). However, they do so upon the restoration of excitatory amino acid agonists such as L-glutamate. [3H]MK-801 provides a molecular probe which is specific for a binding site located within the ionophore of the N-methyl-D-aspartate-type excitatory amino acid receptor, [3H]MK-801 does not bind to non-N-methyl-D-aspartate excitatory amino acid receptors. Exploiting [3H]MK-801 binding as a quantitative measure of agonist activity with respect to ability of inducing the open channel conformation, the present study demonstrates that L-homocysteate is an agonist almost equivalent to L-glutamate in terms of efficacy (maximal N-methyl-D-aspartate response) as well as potency (EC50). The effect of L-homocysteate was dose-dependent, stereospecific (L-homocysteate greater than DL-homocysteate greater than D-homocysteate), suppressible by the N-methyl-D-aspartate-selective competitive antagonist (+/-)-3(2-carboxy-piperazine-4-yl)propyl-l-phosphonate, and potentiated by the N-methyl-D-aspartate-selective "allosteric" modulator glycine. The demonstrated inactivity of L-homocysteine (and virtually all naturally occurring, non-acidic amino acids) implies that the omega-sulphonic acid moiety is an acceptable substitute for the omega carboxyl group for activating the N-methyl-D-aspartate receptor. While the potency of L-homocysteate at N-methyl-D-aspartate receptors was by a factor of only 1.6 smaller than that of L-glutamate, the affinity of L-homocysteate for kainate-type excitatory amino acid receptors was approximately four-fold lower than that of L-glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Maleato de Dizocilpina/metabolismo , Homocisteína/análogos & derivados , Canais Iônicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Neurotransmissores/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Homocisteína/farmacologia , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Cinética , Masculino , Piperazinas/farmacologia , Ratos , Ratos Endogâmicos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores da Fenciclidina , Estereoisomerismo , Estimulação QuímicaRESUMO
Radiation effects are mediated in part by the generation of oxygen-derived free radicals and hydrogen peroxide. Membrane polyunsaturated fatty acids are important biological targets of these toxic molecules which cause lipid peroxidation. Radiation damage to DNA is also known to result in base hydroperoxides, especially thymidine hydroperoxide. Glutathione (GSH) is known to inhibit lipid peroxidation both chemically and through its interaction with the selenium-dependent glutathione peroxidase (GSH-Px). Although cytosolic GSH-Px can metabolize organic lipid peroxides in solution, it cannot metabolize phospholipid peroxides in micelles. This may be due to the interference of phase differences between the aqueous cytosol and the membrane, or the result of steric hindrance. Recent studies have suggested the presence of a membrane-bound GSH-dependent peroxidase system. We examined the cytosolic versus membrane-associated GSH-Px, in various tissues of mice on a selenium and vitamin E deficient diet, and found significant differences among organs in the distribution of enzyme activity in these two subcellular fractions. The effect of single high-dose whole body irradiation did not appear to be related to the activity of these enzymes.
Assuntos
Lesões por Radiação/enzimologia , Selênio/deficiência , Deficiência de Vitamina E/enzimologia , Animais , Medula Óssea/enzimologia , Medula Óssea/efeitos da radiação , Catalase/metabolismo , Citosol/enzimologia , Dieta , Glutationa Peroxidase/fisiologia , Glutationa Transferase/metabolismo , Células-Tronco Hematopoéticas/efeitos da radiação , Mucosa Intestinal/enzimologia , Mucosa Intestinal/efeitos da radiação , Jejuno/efeitos da radiação , Fígado/enzimologia , Masculino , Camundongos , Baço/efeitos da radiação , Frações Subcelulares/enzimologia , Irradiação Corporal TotalRESUMO
Human liver slices, human liver microsomes, and rat liver microsomes were used to investigate the metabolism of 3H-taxol. The effects of drugs frequently coadministered with taxol and the effects of several cytochrome P450 system probes were studied. In all, 16 compounds were screened. After incubation with liver slices or with microsomal protein, 3H-taxol was converted into several radioactive species resolved by HPLC. There were qualitative and quantitative species differences in the metabolism of taxol. The pattern of metabolism was similar for both human-derived preparations, with 6 alpha-hydroxytaxol being the major metabolite peak. In drug interaction studies performed with human liver microsomes, cimetidine 80 microM, and diphenhydramine 200 microM, had little or no effect on 6 alpha-hydroxytaxol formation. Quinidine, ketoconazole, dexamethasone and Cremophor EL inhibited 6 alpha-hydroxytaxol formation with IC50 values of 36 microM, 37 microM, 16 microM and 1 microliter/ml, respectively, but these concentrations exceed the usual clinical range. Cremophor EL also inhibited microsomal metabolism of taxol, but at 2 microliters/ml it had little or no effect on 6 alpha-hydroxytaxol production by human liver slices. These results suggest that: (1) taxol is metabolized by the cytochrome P450 system; (2) taxol metabolism is different in humans than in rats; (3) taxol metabolism in humans is unlikely to be altered by cimetidine, dexamethasone, or diphenhydramine, drugs regularly coadministered with taxol; (4) taxol metabolism can be indirectly affected by Cremophor EL, the formulation vehicle; (5) taxol metabolism may be altered by concentrations of ketoconazole achievable in humans only at very high doses; and (6) taxol metabolism and drug interaction studies of clinical relevance can be performed in vitro with human liver microsomes and human liver slices, but not with rat liver preparations.
Assuntos
Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Taxoides , Animais , Biotransformação , Cimetidina/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Dexametasona/farmacologia , Difenidramina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Glicerol/análogos & derivados , Glicerol/farmacologia , Humanos , Técnicas In Vitro , Cetoconazol/farmacologia , Cinética , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Paclitaxel/análogos & derivados , Paclitaxel/análise , Paclitaxel/metabolismo , Quinidina/farmacologia , Ratos , Especificidade da Espécie , Tensoativos/farmacologia , TrítioRESUMO
The interaction of activated etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), with thiols has been studied both in vitro and in vivo in mice. We have found that both glutathione (GSH) and cysteine rapidly reduce the VP-16 free radical, which results in the regeneration of the parent drug and the oxidation of the thiol. Using spin-trapping and electron spin resonance (ESR) techniques, we have shown that this one-electron/hydrogen donation by thiols forms thiyl radicals (RS.) which are intermediates for the formation of the oxidized thiols. The administration of VP-16 in vivo to mice decreased the total thiol levels in liver and concomitantly increased the formation of oxidized thiols. Furthermore, VP-16 stimulated glutathione reductase in liver. While administration of VP-16 also increased the total thiol pools in kidney, in contrast, no significant effects were observed on lung and heart thiol pools.
Assuntos
Etoposídeo/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Óxidos N-Cíclicos , Cisteína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Glutationa/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , OxirreduçãoAssuntos
Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Hidróxidos/metabolismo , Linhagem Celular , Óxidos N-Cíclicos , Resistência a Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Glutationa Peroxidase/metabolismo , Humanos , Radical Hidroxila , NAD/farmacologia , NADP/farmacologia , Marcadores de SpinAssuntos
Glutationa/análise , Linhagem Celular , Cisplatino/uso terapêutico , Resistência a Medicamentos , Feminino , Glutationa/análogos & derivados , Dissulfeto de Glutationa , Glutationa Peroxidase/análise , Glutationa Redutase/análise , Glutationa Transferase/análise , Humanos , Neoplasias Ovarianas/análise , Neoplasias Ovarianas/enzimologia , Fatores de TempoAssuntos
Adenosina Trifosfatases/metabolismo , Gorduras na Dieta/farmacologia , Mitocôndrias Hepáticas/enzimologia , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Ácidos Dicarboxílicos/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Essenciais/farmacologia , Óleos de Peixe/farmacologia , Masculino , Mitocôndrias Hepáticas/metabolismo , Ratos , Albumina Sérica/farmacologia , Estimulação QuímicaRESUMO
Two cell lines, U-937 and MOLT-4, were used to investigate the toxicity, DNA incorporation, and effect on mitochondria of 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and its putative metabolite 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-uracil (FAU). After 72-hr incubation, the IC50 values for FIAU were 6.4 microM for U-937 cells and 26 microM for MOLT-4 cells. IC50 values for FAU were 10-fold higher in both cell lines. Incubation for 24 hr with 10 microM [2-14C]FIAU led to 2.1% and 0.93% replacement of thymidine in DNA of U-937 and MOLT-4 cells, respectively. The predominant radioactive species measurable in DNA was FIAU. A similar incubation with [2-14C]FAU resulted in 4-fold lower DNA incorporation of a single radioactive species that coeluted with 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). There was no evidence of a selective repair process after DNA incorporation of FIAU or FAU (FMAU). Increased intracellular concentrations of FIAU triphosphate and incorporation into DNA were associated with an increase in cellular toxicity. Continuous exposure to a clinically achievable concentration of FIAU, 0.44 microM, produced a constant DNA incorporation of 0.80% and 0.11% for U-937 and MOLT-4 cells, respectively. FIAU was not readily metabolized to FAU or iodouracil by human liver in vitro. Compared with 2',3'-dideoxycytidine as a positive control, after 12 days of continuous exposure of U-937 and MOLT-4 cells to FIAU there was no evidence of increased lactate production. These data negate several possible mechanisms (DNA chain termination, DNA polymerase inhibition, one form of selective mitochondrial poisoning, and FAU-mediated toxicity) and provide clues for possible mechanisms (FIAU triphosphate concentration and DNA incorporation). Further work is needed to develop a complete explanation for the delayed hepatic toxicity observed in the investigational clinical trials of FIAU.
Assuntos
Arabinofuranosiluracila/análogos & derivados , Reparo do DNA , DNA/metabolismo , Lactatos/metabolismo , Arabinofuranosiluracila/metabolismo , Arabinofuranosiluracila/toxicidade , Humanos , Fígado/metabolismo , Estrutura Molecular , Células Tumorais CultivadasRESUMO
The present investigation attempted to differentiate haloperidol-sensitive sigma sites (sigma H) from phencyclidine (PCP) binding sites in rat brain membranes. We studied the effects of several cations at physiologically relevant concentrations on the binding of radioligands selective for sigma H sites ([3H]haloperidol, [3H](+)3-PPP**), and [3H](+)SKF10,047), or for PCP sites ([3H]PCP and [3H]TCP). The PCP sites displayed a markedly greater sensitivity to cations than sigma H sites. This property was reflected by a greater extent of inhibition of the binding of PCP-selective relative to sigma H-selective ligands at a given cation concentration, as well as by lower IC50's and by steeper slopes of the cation dose-response curves. Divalent cations were approximately 100 times more potent than monovalent cations. All cations were inhibitory, except Sr2+ and Ba2+ which, at micromolar concentrations, enhanced PCP binding but not sigma H binding. Thus, PCP-selective sites appeared to be distinct from sigma H sites with regards to several aspects of cation modulation. This is consistent with the view that PCP and sigma H sites are distinct molecular entities. Further, the marked cation sensitivity of the PCP site is consistent with the current hypothesis according to which the PCP site is linked to the N-methyl-D-aspartate (NMDA) receptor-cation channel complex.
Assuntos
Encéfalo/ultraestrutura , Cátions/farmacologia , Fenciclidina/metabolismo , Fator sigma/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Haloperidol/farmacologia , Ligantes , Masculino , Fenazocina/análogos & derivados , Fenazocina/metabolismo , Ratos , Ratos Endogâmicos , TrítioRESUMO
Adriamycin-stimulated formation of .OH in sensitive and resistant subline of human breast tumor cells (MCF-7) has been examined by electron spin resonance spectroscopy. It was shown that adriamycin significantly stimulated the formation of .OH spin adducts [5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH] in the sensitive cells but not in the resistant cells. By use of spin-broadening techniques and inhibition of .OH with high molecular weight poly(ethylene glycol), which does not enter intact cells, it was shown that 60-65% of adriamycin-induced .OH were located extracellularly and were metal ion dependent since they were decreased in the presence of desferal. Furthermore, superoxide dismutase and catalase, enzymes that detoxify superoxide and hydrogen peroxide, also significantly inhibited adriamycin-induced .OH formation and protected against the cytotoxicity of adriamycin. The differential .OH formation in these two cell lines is not due to diminished activities of flavin-dependent activating enzymes nor decreased accumulation of the drug in the cells but appears to be related to enhanced activities of detoxifying enzymes, particularly, glutathione peroxidases in the resistant cells.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ensaio de Unidades Formadoras de Colônias , Doxorrubicina/farmacologia , Ensaio Tumoral de Célula-Tronco , Neoplasias da Mama/enzimologia , Catalase/fisiologia , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Desferroxamina/farmacologia , Resistência a Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Radicais Livres , Glutationa Peroxidase/fisiologia , Humanos , Hidróxidos/biossíntese , Radical Hidroxila , Oxigênio/metabolismo , Superóxido Dismutase/fisiologiaRESUMO
1. The main metabolites of rifampin and rifabutin in man are their respective 25 deacetylated derivatives, but the enzyme(s) responsible for these biotransformations are not known. 2. In experiments with human liver slices and human liver microsomes, the 25 deacetylated derivatives of these drugs were the main metabolites observed. Slices and microsomes metabolized rifabutin 3-6-fold faster than rifampin, in agreement with their relative clearance in patients. Rifabutin partitioned into slices more avidly than rifampin. 3. In microsomal incubations, deacetylation did not require NADPH, but the amount of metabolite at the end of incubation was affected by NADPH. With NADPH the amount of 25 deacetyl rifabutin decreased, whereas the amount of 25 deacetyl rifampin increased slightly. A panel of liver microsomes from seven donors showed a 3-4-fold difference in the formation of 25 deacetyl rifabutin or 25 deacetyl rifampin, with strong correlation between the production of the two metabolites (r2 = 0.94). 4. The production of 25 deacetyl rifabutin and 25 deacetyl rifampin by human liver microsomes was not significantly affected by 1 microM 4 chloromercuricbenzoic acid or bis-(4-nitrophenyl) phosphate, but was completely inhibited by 1 microM paraoxon or 1 microM diisopropylfluorophosphate. These results indicate that in man rifampin and rifabutin are deacetylated to their main metabolites by B-esterases.
Assuntos
Antibióticos Antituberculose/farmacocinética , Esterases/metabolismo , Fígado/enzimologia , Rifabutina/farmacocinética , Rifampina/farmacocinética , Acetilação , Antibióticos Antituberculose/metabolismo , Biotransformação , Humanos , Microssomos Hepáticos/enzimologia , NADP/metabolismo , Rifabutina/metabolismo , Rifampina/metabolismoRESUMO
Adriamycin-resistant (AdrR) human breast cancer cells have been selected which exhibit cross-resistance to a wide range of anti-cancer drugs. This multidrug-resistant phenotype is associated with increases in the activities of glutathione peroxidase and glutathione transferase. The 45-fold increase in glutathione transferase activity is associated with the appearance of a new anionic isozyme in AdrR cells which is immunologically related to the anionic glutathione transferase present in human placenta. The increase in transferase and the level of drug resistance is relatively stable during passage of AdrR cells in the absence of adriamycin for over 10 months. A similar anionic glutathione transferase isozyme is also found in rat hyperplastic liver nodules, a preneoplastic state resulting from exposure to carcinogens. A rat cDNA which codes for the anionic glutathione transferase in rat hyperplastic nodules hybridizes to a 1.1-kilobase pair mRNA which is overexpressed in the AdrR MCF-7 cells. The anionic transferase has been purified from the AdrR cells and found to have characteristics which distinguish it from other anionic human glutathione transferases, including high levels of intrinsic peroxidase activity. The overexpression of a similar anionic glutathione transferase in human breast cancer cells selected for multidrug resistance and in rat hyperplastic liver nodules, which develop resistance to various hepatotoxins, suggests a possible role for this drug-conjugating enzyme in the mechanism of resistance in both of these states.