RESUMEN
Antisense oligonucleotides can vary significantly and unpredictably in their ability to inhibit protein synthesis. Libraries of chimeric oligonucleotides and RNase H were used to cleave and thereby locate sites on human multidrug resistance-1 RNA transcripts that are relatively accessible to oligonucleotide hybridization. In cell culture, antisense sequences designed to target these sites were significantly more active than oligonucleotides selected at random. This methodology should be generally useful for identification of potent antisense sequences. Correlation between oligonucleotide activity in the cell culture assay and in an in vitro RNase H assay supports the proposed role of the enzyme in the mechanism of antisense suppression in the cell.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Biblioteca de Genes , Oligonucleótidos Antisentido/análisis , ARN Mensajero/análisis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/química , Rodaminas/metabolismo , Ribonucleasa H/metabolismo , Células Tumorales CultivadasRESUMEN
A series of nitrocoumarin and nitrochromene derivatives have been prepared and shown to inhibit the phosphatidylinositol-specific phospholipase C(PLC)(IC50 < 10 micrograms/mL) isolated from human melanoma. The inhibition of PLC by nitrocoumarin 4a was time-dependent and irreversible. The inhibition of PLC was shown to interfere with inositide metabolism in whole cells (IC50 = 4 micrograms/mL) in a manner consistent with their proposed mode of activity. Finally, the compounds were shown to be growth inhibitory to cultured melanoma cells (ID50 = 2 micrograms/mL), suggesting that PLC may be an attractive new target for chemotherapeutic intervention.
Asunto(s)
Antineoplásicos/síntesis química , Cromonas/síntesis química , Cumarinas/síntesis química , Fosfolipasas de Tipo C/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Humanos , Melanoma/enzimología , Ratones , Fosfatidilinositoles/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
(R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]- bis[5-nitro-1H-benz[de]isoquinoline-1,3-(2H)-dione] dimethanesulfonate (DMP 840), is a bis-naphthalimide anticancer tumoricidal agent currently in phase I clinical trials. DMP 840 exhibits curative activity in human tumor xenografts, where it shows selectivity for human solid tumors over murine leukemias. In contrast to the selectivity found for DMP 840 in vivo, DMP 840 exhibits potent antiproliferative activity in vitro against a variety of human and murine leukemia and solid tumor cell lines in culture, with inhibitory doses that reduce the number of treated cells to one half (IC50) values ranging from 2.3 to 53 nM. DMP 840 was growth inhibitory to three doxorubicin-resistant cell lines with IC50 values also in the nanomolar range. Clonogenic survival experiments showed that DMP 840 was equally cytotoxic to both exponentially growing and quiescent human clone A colon carcinoma cells. A 1-h incubation of DMP 840 (1.22-12 microM) caused 5-log cell kill in KB-3-1 human epidermoid carcinoma, clone A human colon carcinoma, and L1210 murine leukemia cell lines. The rapid cell killing by DMP 840 in clonogenic survival experiments and initial mechanism of action studies was consistent with a DNA-interactive mechanism for DMP 840 cytotoxicity. Mechanism of action studies in L1210 leukemia cells demonstrated that DMP 840 inhibited the incorporation of thymidine and uridine into DNA and RNA with IC50 values of 0.55 and 0.08 microM, respectively. DMP 840 produced DNA single-strand breaks in a dose-dependent manner. Double-strand breaks were not observed with DMP 840 treatment, even at higher concentrations of compound. Chinese hamster ovary (CHO) and P388 cells resistant to camptothecin and containing a mutant form of topoisomerase I were also used to evaluate whether DMP 840 was cross-resistant with agents active against topoisomerase I. While the CHOR line was 163-fold resistant to camptothecin, the CHOR line was only 1.7-fold resistant to DMP 840. In summary, DMP 840 is a DNA-interactive agent that demonstrates excellent antiproliferative activity in vitro against cultured tumor cells from both human and murine sources. Its mechanism of tumoricidal activity may be novel.
Asunto(s)
Antineoplásicos/toxicidad , Isoquinolinas/toxicidad , Mesilatos/toxicidad , Amsacrina/toxicidad , Animales , División Celular/efectos de los fármacos , Ensayos Clínicos Fase I como Asunto , Neoplasias del Colon , Dactinomicina/toxicidad , Relación Dosis-Respuesta a Droga , Doxorrubicina/toxicidad , Resistencia a Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células KB , Cinética , Leucina/metabolismo , Leucemia L1210 , Leucemia P388 , Neoplasias Mamarias Experimentales , Melanoma , Melanoma Experimental , Ratones , Mitoxantrona/toxicidad , Timidina/metabolismo , Trasplante Heterólogo , Células Tumorales Cultivadas , Uridina/metabolismoRESUMEN
We have synthesized a promising class of bis-naphthalimide anti-tumor agents. A representative compound in this series, XB596, exhibits potent in vitro growth inhibitory activity against several human and murine leukemic and solid tumor lines in culture, with IC50 values ranging from 7.2 to 147.5 nM. XB596 was almost as equally growth inhibitory against three doxorubicin-resistant cell lines compared with their parental lines. Using a human tumor colony-forming assay, XB596 demonstrated cytocidal activity against fresh human tumors taken directly from patients, with 23 of 25 evaluable tumors responding to a continuous exposure of 1 microgram/ml of XB596. When L1210 cells were incubated with XB596 for 1 h, the incorporation of uridine and thymidine into RNA and DNA, respectively, was inhibited with IC50 values of 0.14 microM. DNA single-strand breaks, but not double-strand breaks, were detected in XB596-treated L1210 cells. XB596 bound to DNA with guanine-cytosine sequence selectivity as shown by an indirect ethidium bromide displacement assay. XB596 was shown to interact with DNA by a spectrophotometric titration assay, with an estimated binding constant of 4.7 +/- 2.2 +/- 10(6) M-1. XB596 unwound supercoiled DNA as measured by agarose gel electrophoresis. These data are consistent with XB596 being a DNA intercalator. In vivo, XB596 demonstrated good anti-tumor activity against two human solid tumors (DLD-2 colon adenocarcinoma and MX-1 mammary carcinoma) xenografted in nude mice, but has not demonstrated anti-leukemic activity. In summary, XB596 is a pre-clinical anti-cancer agent which interacts with DNA and demonstrates good in vivo anti-tumor activity against human solid tumor xenografts.
Asunto(s)
Antineoplásicos/farmacología , Naftalenos/farmacología , Propilaminas/farmacología , Animales , División Celular/efectos de los fármacos , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , Humanos , Leucemia L1210/tratamiento farmacológico , Leucemia P388/tratamiento farmacológico , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias Experimentales/tratamiento farmacológico , Trasplante Heterólogo , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
The novel anticancer drug candidate brequinar sodium (DuP 785, NSC 368390, 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline- carboxylic acid sodium salt) was shown previously to be an inhibitor of dihydroorotate dehydrogenase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway. Brequinar sodium inhibits the activity of this enzyme isolated from mammalian sources only but not those forms isolated from yeast or bacteria, which also use ubiquinone as the cofactor. Brequinar sodium also does not inhibit the activity of a soluble Zymobacterium oroticum dihydroorotate dehydrogenase which uses NAD+ as a cofactor. Brequinar sodium inhibits L1210 dihydroorotate dehydrogenase with mixed inhibition kinetics with respect to either the substrate (dihydroorotate) or the cofactor (ubiquinone Q6) with Ki' values in the 5-8 nM range. Our results suggest that brequinar sodium inhibits dihydroorotate dehydrogenase by binding to the enzyme at a unique site that is distinct from the dihydroorotate or the ubiquinone-binding site. This binding site appears to be unique to the mammalian enzyme, because brequinar sodium does not inhibit the yeast, Escherichia coli, or Z. oroticum forms of the enzyme.
Asunto(s)
Antineoplásicos/farmacología , Compuestos de Bifenilo/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Animales , Dihidroorotato Deshidrogenasa , Cinética , Leucemia L1210/enzimología , Ratones , Naftoquinonas/farmacología , Ácido Orótico/farmacologíaRESUMEN
The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.
Asunto(s)
Butiratos/farmacología , Glioma/análisis , Glicoproteínas/análisis , Activadores Plasminogénicos/análisis , Animales , Ácido Butírico , Relación Dosis-Respuesta a Droga , Glioma/genética , Glioma/patología , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Activadores Plasminogénicos/antagonistas & inhibidores , Inactivadores Plasminogénicos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The distribution of the novel anticancer drug candidate Brequinar Sodium (DuP 785, NSC 368390) was studied in control mice and mice implanted subcutaneously with human colon carcinoma xenografts. Mice were given radiolabeled 14C-Brequinar Sodium intravenously. Brequinar concentrations in blood and various tissues were determined at 1, 6, and 24 h after drug administration. Within 1 h Brequinar distributed to the tumor and all other tissues studied. The tumor-to-blood drug concentration ratios ranged from 0.19 to 0.41. Radioactivity in the liver and small intestine at 1 h accounted for 17% and 13%, respectively, of the dose given. Elimination rates of Brequinar from all tissues were approximately equal to that from blood. Comparison of blood concentrations determined by both radioactivity and HPLC methods suggests that the intact drug is probably the only form in the blood.