RESUMEN
Interferons (IFN) are biologic agents involved in the antiviral response and the inhibition of tumor growth. Biochemical pathways of IFN action include the double-stranded RNA-activated oligoadenylate synthetase, RNase L, and double-stranded RNA-dependent protein kinase (PKR). Extracellular ribonucleases, especially onconase, also display antiviral and antitumor properties and involve degradation of RNA. We find that IFN increases the anticancer activity of onconase. These two agents work synergistically, and the effect is seen at the level of translation probably because of the degradation of tRNA.
Asunto(s)
Antineoplásicos/farmacología , Proteínas del Huevo/farmacología , Interferones/farmacología , Ribonucleasas/farmacología , Animales , Sinergismo Farmacológico , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/enzimología , Modelos Logísticos , Biosíntesis de Proteínas/efectos de los fármacos , Rana pipiens , Células Tumorales CultivadasRESUMEN
PURPOSE: To examine the histopathology of the kidney in mice following repeated injections of the antitumor drug onconase, and to determine whether lysine, which reportedly blocks kidney uptake of other basic proteins, blocks the high renal uptake of onconase. METHODS: Mice received repeated intraperitoneal onconase injections over 3 weeks. Kidneys were examined by light microscopy after 1 week, 3 weeks, and 5 weeks (2 weeks after cessation of injections) and compared to kidneys from animals receiving a similar schedule of PBS injections. Renal uptake of radioiodinated onconase was measured in animals receiving intraperitoneal injections of lysine solutions of acidic and neutral pH given at -30, 0 and + 5 min relative to intravenous onconase injection. Renal onconase uptake was also measured in animals made metabolically acidotic by ingestion of ammonium chloride, arginine chloride or lysine dihydrochloride from the drinking water. RESULTS: Onconase caused acute moderate multifocal proximal renal tubular necrosis, and this toxicity was reversed by 2 weeks after drug withdrawal. Intraperitoneal injections of lysine dihydrochloride in PBS (pH 1.5) reduced renal onconase uptake at 15 min from 67.9+/-13.4% to 17.0+/-3.8% of the injected dose without affecting the plasma concentration and also reduced the fraction of degraded onconase in the urine. However, neutral solutions of lysine dihydrochloride at pH 7.4 or lysine acetate at pH 7.1 were ineffective at blocking renal onconase uptake. Furthermore, renal onconase uptake was minimally or not affected by a state of metabolic acidosis. CONCLUSIONS: Proximal tubular toxicity of onconase was reversible. Renal onconase uptake was blocked by lysine at pH 1.5 but not at neutral pH.
Asunto(s)
Antineoplásicos/toxicidad , Proteínas del Huevo/toxicidad , Riñón/efectos de los fármacos , Lisina/farmacología , Ribonucleasas/toxicidad , Acidosis/metabolismo , Animales , Proteínas del Huevo/farmacocinética , Femenino , Concentración de Iones de Hidrógeno , Riñón/metabolismo , Riñón/patología , Ratones , Ratones Endogámicos BALB C , Ribonucleasas/farmacocinéticaRESUMEN
Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.
Asunto(s)
Proteínas/genética , Ribonucleasa Pancreática/genética , Ribonucleasas/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Neurotoxina Derivada del Eosinófilo , Glioma/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Leucina/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Plásmidos , Ingeniería de Proteínas , Proteínas/farmacología , Receptores de Transferrina/inmunología , Proteínas Recombinantes/metabolismo , Ribonucleasa Pancreática/farmacología , Porcinos , Transferrina/uso terapéutico , Células Tumorales CultivadasRESUMEN
The similarities and differences among members of the RNase A superfamily provide an ideal opportunity to examine the molecular basis for differences in their pharmacokinetics and biodistribution. Plasma clearances in BALB/c mice are similar among the five RNases studied: human pancreatic RNase, angiogenin, eosinophil-derived neurotoxin, onconase, and bovine seminal RNase. The average clearance is 0.13 ml/min or 60% of the glomerular filtration rate (measured by [14C]inulin clearance during continuous infusion from an i.p. implanted osmotic pump). Angiogenin has a higher volume of distribution and plasma-to-muscle transport rate than the other RNases, suggestive of binding to endothelial cells. Organ distribution differs dramatically among these RNases. The RNase most toxic to tumor cells, onconase, exhibits the longest retention in the kidneys: at 180 min, 50% of the injected dose is found in the kidneys, whereas only 1% or less of the other RNases is retained in the kidneys. Slower elimination of onconase from the kidneys may be due to a higher degree of binding in the kidney or a resistance to proteolytic degradation. To elucidate the molecular determinants involved in tissue uptake, we examined the biodistribution of recombinant onconase and two onconasepancreatic RNase chimeric proteins. The tissue retention property of onconase appears to be located in at least two regions, one of which is in the NH2-terminal 9-amino acid alpha-helix. The NH2-terminal pyroglutamate of onconase, a residue essential for ribonucleolytic activity and cytotoxicity, does not play a role in kidney retention.
Asunto(s)
Ribonucleasa Pancreática/farmacocinética , Ribonucleasas/farmacocinética , Animales , Bovinos , Femenino , Tasa de Filtración Glomerular , Humanos , Infusiones Parenterales , Inulina/farmacocinética , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Óvulo/enzimología , Proteínas/farmacocinética , Rana pipiens , Ribonucleasa Pancreática/sangre , Ribonucleasa Pancreática/aislamiento & purificación , Ribonucleasas/sangre , Ribonucleasas/aislamiento & purificación , Semen/enzimología , Vesículas Seminales/enzimología , Factores de Tiempo , Distribución TisularRESUMEN
A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Proteínas del Huevo/metabolismo , Inhibidores Enzimáticos/farmacología , Hormonas Placentarias/farmacología , Ribonucleasa Pancreática/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas del Huevo/antagonistas & inhibidores , Proteínas del Huevo/química , Proteínas del Huevo/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis , Concentración Osmolar , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/metabolismo , ARN/metabolismo , Rana pipiens , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa Pancreática/antagonistas & inhibidores , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/farmacología , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/química , Ribonucleasas/farmacología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Effect of monensin, intercalated in liposomes on the cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in phagocytic and non-phagocytic cells as well as in mice has been studied. Intercalation does not disturb the integrity of the liposomal bilayer and substantially enhances the cytotoxicities of ricin and Pseudomonas exotoxin A in both phagocytic and non-phagocytic cells while it has no effect on diphtheria toxin. The observed effect is highly dependent on the liposomal lipid composition as well as cell types. The potentiating ability of monensin in neutral vesicle is 2.2-fold higher than in negatively charged vesicles in non-phagocytic cells while no difference was observed in phagocytic cells. Incorporation of stearylamine in liposomes reduces the potentiating effect of monensin. Liposomal monensin has also been found to enhance the cytotoxicity of ricin in mouse in vivo in a dose-dependent manner and is maximal when ricin is injected within 60 min of monensin injection. Liposomal monensin remains in circulation for 2 hr while free monensin remains only for 15 min. Tissue distribution studies reveal that liposomal monensin is present mainly in the liver and spleen which are also the major sites for ricin accumulation. Thus liposome is found to be an effective delivery vehicle for monensin to potentiate the cytotoxicity of immunotoxins or hormonotoxins and could prove useful for selective elimination of cancer cells.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Supervivencia Celular/efectos de los fármacos , Monensina/toxicidad , Ricina/toxicidad , Factores de Virulencia , Animales , Células CHO , Cricetinae , Toxina Diftérica/toxicidad , Portadores de Fármacos , Sinergismo Farmacológico , Exotoxinas/toxicidad , Membrana Dobles de Lípidos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Monensina/administración & dosificación , Fagocitosis , Pseudomonas aeruginosa , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Monensin, a carboxylic ionophore, which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8-20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.
Asunto(s)
Monensina/farmacología , Ricina/toxicidad , Animales , Dextranos , Portadores de Fármacos , Sinergismo Farmacológico , Fluoresceína-5-Isotiocianato , Liposomas/metabolismo , Masculino , Ratones , Monensina/administración & dosificación , Monensina/farmacocinética , Análisis de Supervivencia , Distribución TisularRESUMEN
A complement-mediated liposome immune lysis assay using entrapped calcein was developed for a plant toxin gelonin. Gelonin was covalently coupled to DPPE, and then adsorbed on to the surface of liposomes. Such antigen-bearing liposomes when incubated with anti-gelonin antibody in the presence of guinea pig complement undergo lysis. The detection range is from 3 ng to 60 ng. The method was used to monitor isolation of gelonin by affinity chromatography. It was observed that a minor peak in addition to the major one comes with gelonin, shared common epitopes/epitope with gelonin in immunological reaction. This was further confirmed by SDS-gel electrophoresis indicating the former being an isoform of gelonin. A comparative study of the immunocross-reactivity of ricin and ricin A chain with anti-gelonin antibody was carried out. It was found that while ricin A chain cross-reacted extensively with gelonin antibody and intact ricin elicited little or no cross-reactivity. It is suggested that the present LILA may be employed for the detection and quantitation of ricin A chain by this LILA method.