RESUMEN
DNA single-strand and double-strand breaks produced by doxorubicin and two anthracycline derivatives (4-demethoxy-daunorubicin and 4'-deoxy-4'-iododoxorubicin) were measured in doxorubicin-sensitive and -resistant P388 leukemia cell lines, using filter elution methods, and compared with cellular drug accumulation to account for major differences in their cytotoxic activities and cross-resistance. The increased cytotoxic potency of the two derivatives reflects at least in part the enhanced drug accumulation by cells that results from their increased lipophilicity. However, the level of protein-linked DNA breaks was not directly related to cellular accumulation of drug analogues. It is possible that enhanced cytotoxicity may also be the consequence of the greatly enhanced ability of analogues to cause DNA strand breaks. The resistant line showed only a modest degree of resistance to both anthracycline derivatives compared with the high degree of resistance to doxorubicin. Although for all the anthracyclines tested drug accumulation was reduced in the resistant line, this did not correlate with the degree of resistance. A differential sensitivity of resistant and parental cell lines to DNA cleavage activity was consistently found for all three drugs tested. However, in contrast to a lack of effect of doxorubicin, the derivatives caused appreciable DNA strand breakage in resistant cells. The enhanced ability of these analogues to break DNA in resistant cells is consistent with the slight cross-resistance with doxorubicin. DNA double-strand breaks produced in isolated nuclei from these cells paralleled the pattern found in whole cells, thus indicating that a nuclear alteration, presumably involving DNA topoisomerases, is associated with anthracycline resistance. Our findings strongly support the hypothesis that anthracycline resistance in these cell variants may be mediated by multiple mechanisms, involving alterations of plasma membrane and changes of nuclear enzymatic activities responsible for DNA strand breaks.
Asunto(s)
ADN/efectos de los fármacos , Leucemia P388/genética , Leucemia Experimental/genética , Animales , Antibióticos Antineoplásicos , Línea Celular , Núcleo Celular/efectos de los fármacos , Daunorrubicina/análogos & derivados , Daunorrubicina/uso terapéutico , Doxorrubicina/análogos & derivados , Doxorrubicina/uso terapéutico , Idarrubicina , Leucemia P388/tratamiento farmacológico , Ratones , Naftacenos/uso terapéutico , Conformación de Ácido Nucleico/efectos de los fármacosRESUMEN
Four new derivatives of daunorubicin and two new derivatives of Adriamycin characterized by the absence of the methoxyl groups at the C-4 position have been studied in cell cultures in vitro to establish structure-activity relationships. 4-Demethoxydaunorubicin was 27 to 100 times more active than was daunorubicin when inhibiting the cloning efficiency of exponential-phase HeLa cells and, like daunorubicin, was slightly active on early plateau-phase cells. DNA synthesis in mouse embryo fibroblasts stimulated by fetal calf serum was inhibited equally by the two compounds, although 4-Demethoxydaunorubicin was slightly more active than was daunorubicin when inhibiting RNA synthesis. The beta anomer of 4-demethoxydaunorubicin showed a reduced activity on HeLa cells compared to its alpha anomer, but it was equally active on DNA synthesis. The stereoisomers of 4-demethoxydaunorubicin bearing the inverted configuration in positions 7 and 9 were devoid of significant cytotoxic activity and were only slightly active on DNA synthesis at the doses tested. 4-demethoxyadriamycin and 4-demethoxy-4'-epi-adriamycin were 65 to 500 times more active than was Adriamycin on HeLa cell cloning efficiency and about 10 times more active on DNA synthesis in mouse embryo fibroblasts. Cell uptake in mouse embryo fibroblasts was also investigated for all the new derivatives tested.
Asunto(s)
ADN/biosíntesis , Daunorrubicina/análogos & derivados , Doxorrubicina/análogos & derivados , ARN/biosíntesis , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Daunorrubicina/metabolismo , Daunorrubicina/farmacología , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Relación Estructura-Actividad , Timidina/metabolismo , Uridina/metabolismoRESUMEN
The synthesis of 4'-epi-daunorubicin and of 4'-epi-adriamycin was performed by condensation of 2,3,6-trideoxy-3-trifluoroacetamido-4-O-trifluoroacetyl-alpha-L-arabino-hexopyranosyl chloride with daunomycinone or the protected adriamycinone derivative 17, respectively. Both the alpha and beta anomers were obtained and characterized. All new compounds are biologically active in cultured cells and the alpha anomers display noticeable activity in experimental tumors in mice. Interestingly, 4'-epi-adriamycin (4) appears nontoxic to cultured heart cells up to a concentration of 5 mug/ml.
Asunto(s)
Aminoglicósidos/síntesis química , Antineoplásicos/síntesis química , Daunorrubicina/análogos & derivados , Doxorrubicina/análogos & derivados , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Animales , Antineoplásicos/farmacología , Células Cultivadas , Daunorrubicina/síntesis química , Daunorrubicina/uso terapéutico , Doxorrubicina/síntesis química , Doxorrubicina/uso terapéutico , Células HeLa/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Leucemia Experimental/tratamiento farmacológico , Ratones , Virus de la Leucemia Murina de Moloney/efectos de los fármacos , Sarcoma 180/tratamiento farmacológico , Virus del Sarcoma Murino/efectos de los fármacos , EstereoisomerismoRESUMEN
Phosphatidylinositol added to the medium markedly stimulated the growth-promoting effect of mitogens in normal cells (human lymphocytes and mouse embryo fibroblasts). However, it did not significantly affect quiescent cells or proliferating tumor cell lines (HeLa and MCF-7). The results are consistent with the suggested role of phosphatidylinositol in the widespread mechanism of calcium mobilization.
Asunto(s)
Mitógenos/farmacología , Fosfatidilinositoles/farmacología , Adenocarcinoma/fisiopatología , Animales , Neoplasias de la Mama/fisiopatología , Línea Celular , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Células HeLa/efectos de los fármacos , Células HeLa/fisiología , Humanos , Cinética , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Ratones , Ratones Endogámicos BALB CRESUMEN
It has been demonstrated previously that neoplastic cells with reduced oxygen consumption are more sensitive to doxorubicin8. We have examined the relationship between doxorubicin sensitivity and oxygen consumption of P388 murine leukaemia cell line (P388) and of a doxorubicin resistant subline (P388/dx). Oxygen utilization by P388/dx cells was higher than that found in the sensitive line. A variety of calcium antagonists, including channel blockers and intracellular antagonists (verapamil, trifluoperazine, dantrolene, TMB-8, nitrendipine) or membrane acting drugs (lucensomycin), enhanced the cytotoxic activity of doxorubicin in P388 and markedly in P388/dx subline. This action was accompanied by a reduction of oxygen consumption more pronounced in the resistant cells. These findings emphasizé the correlation between oxygen uptake, instead of calcium dependent processes, and doxorubicin responsiveness. The calcium ionophores A 23187 failed to alter doxorubicin activity in P388 and P388/dx leukaemia.
Asunto(s)
Leucemia P388/tratamiento farmacológico , Leucemia Experimental/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos , Calcimicina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Doxorrubicina/uso terapéutico , Interacciones Farmacológicas , Lucensomicina/farmacología , Ratones , Naftacenos/uso terapéutico , Consumo de Oxígeno/efectos de los fármacosRESUMEN
In order to investigate the involvement of Protein Kinase C (PKC) in the signal transduction mechanisms related to intrinsic chemoresistance, two cellular clones were isolated from LoVo/WT colon adenocarcinoma cell line and their cytogenetic pattern was studied: LoVo C1.7 was intrinsically resistant to Doxorubicin while LoVo C1.5 showed the same resistance index as the mixed parental cell population. Two PKC isoforms, immunologically identified as beta and alpha PKC, were isolated from the cytosolic fraction of all cell types and one single peak of alpha PKC was obtained from the particulate fraction. Resistant LoVo C1.7 cells showed a significant increase of PKC activity; preincubation with H-7 induced PKC inhibition and reversal of drug resistance. These data suggest that in our cell system the identified calcium-dependent PKC subtypes can play a role in the mechanisms of intrinsic resistance.
Asunto(s)
Calcio/farmacología , Doxorrubicina/toxicidad , Resistencia a Medicamentos , Isoenzimas/metabolismo , Isoquinolinas/farmacología , Piperazinas/farmacología , Proteína Quinasa C/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Adenocarcinoma , Supervivencia Celular/efectos de los fármacos , Cromatografía , Aberraciones Cromosómicas , Neoplasias del Colon , Citosol/enzimología , Durapatita , Humanos , Hidroxiapatitas , Isoenzimas/antagonistas & inhibidores , Isoenzimas/aislamiento & purificación , Cariotipificación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/aislamiento & purificación , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
The results of a study of the effects of hydroxyl groups at positions, 2, 4 and 6 of the amino sugar on the activity of daunorubicin, adriamycin, and stereoisomers are presented. While the 4'-deoxy derivatives showed a slightly increased biological activity as compared with the parent compounds, the derivatives containing an additional hydroxyl group were less active. It is suggested that the changes in the polarity and in the DNA binding ability of these derivatives are the main factors accounting for the difference in the in vivo activity. The possible relations among the pKa values, the DNA binding properties, and the cellular uptake of the compounds are discussed with particular reference to their therapeutic effectiveness.
Asunto(s)
Daunorrubicina/análogos & derivados , Doxorrubicina/análogos & derivados , Animales , Sitios de Unión , Células Clonales , ADN/metabolismo , Daunorrubicina/farmacología , Daunorrubicina/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Células HeLa/efectos de los fármacos , Leucemia L1210 , Leucemia Experimental/tratamiento farmacológico , Ratones , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The pathogenesis of celiac disease is not completely understood but, although the initial step of the process is still unclear, an altered immune response seems to play a major role. Previous studies of the biological properties of gliadin have highlighted its cytotoxic effects, and the aim of this study was to develop an in vitro technique to study them. The LoVo (human colon adenocarcinoma) cell line grown in two-dimensional cultures was exposed to different concentrations of digested bread wheat gliadin (62, 125, 250, 500 and 750 microg/ml) for 48 h, after which cell growth and oxidative balance (the content of reduced glutathione (GSH), and peroxidase, transferase and reductase activity) was evaluated. Other food proteins were used as controls. Our data revealed a statistically significant inhibition of cell growth in proportion to the gliadin concentration (from 26 to 100%), combined with a decrease in GSH content (-38% at 500 microg/ml) and reduced enzymatic activity (-30% at 500 microg/ml). The controls did not show any noxious effect. Our results confirm the usefulness of LoVo cells in evaluating gliadin cytotoxicity and that they can be used to investigate the biological properties of gliadin.
Asunto(s)
Adenocarcinoma/patología , Enfermedad Celíaca/fisiopatología , Neoplasias del Colon/patología , Gliadina/efectos adversos , División Celular , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Humanos , Oxidorreductasas/farmacología , Peroxidasa/farmacología , Transferasas/farmacología , Células Tumorales CultivadasRESUMEN
The ultrastructural evaluation of the early alterations adriamycin-induced on cultured mice heart cells is reported. The major effects are hypertrophy of the sarcoplasmic reticulum and a market increase of the number and total extension of the gap junctions. These findings are discussed in the light of the information available in the literature.
Asunto(s)
Doxorrubicina/efectos adversos , Corazón/efectos de los fármacos , Miocardio/ultraestructura , Transporte Biológico/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Desmosomas/efectos de los fármacos , Desmosomas/ultraestructura , Doxorrubicina/farmacología , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/ultraestructura , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/ultraestructuraRESUMEN
Doxorubicin tested at the concentration of 1-2 X 10(-7)M inhibited the cloning efficiency of MS2T cells following 22 and 48 h exposure in complete medium. In the same experimental conditions the [3H]thymidine incorporation was practically unaffected. The inhibitory effect of doxorubicin on cloning efficiency appeared to be directly related with the serum concentration. In fact, this effect became more marked when the cloning efficiency was stimulated by increasing serum concentration in the cultural medium. However, this effect did not seem to be Ca2+ dependent. Similarly doxorubicin displayed a strong inhibitory effect, when the proliferative activity was stimulated by an optimal combination of cAMP and low Ca2+. On the contrary the inhibitory effect of doxorubicin was markedly reduced when the Ca2+ concentration reached the physiological value. These results confirm the direct correlation of the killing effect of doxorubicin with proliferative activity of the cells.
Asunto(s)
Calcio/farmacología , División Celular/efectos de los fármacos , AMP Cíclico/farmacología , Doxorrubicina/farmacología , Animales , Sangre , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Interacciones Farmacológicas , Células HeLa , Ratones , Ratones Endogámicos BALB C , Sarcoma Experimental , Timidina/metabolismoRESUMEN
Two cell lines derived from a primary MSV-M-induced tumor in a BALB/c mouse were studied. One line (MS-2) was subject only to continuous tissue culture transfer (tct). After 21 tct, MS-2 cells produced progressive tumors (MS-2 tumors) in syngeneic hosts. The second cell line (MS-2T) was established by cultivation of a MS-2 tumor. The ability to produce progressive tumors decreased with increased number of tct, in both cell lines. The virus content of MS-2 and MS-2T cells was very low, as shown by uridine incorporation and electron microscopy. Immmunofluorescence tests demonstrated that antigens different from the viral MSV-M antigens were present on the cell lines, and that antigenic changes occurred with increased number of tct. Serum of mice bearing progressive MS-2 tumors reacted with MS-2T cells when these cells produced progressive tumors and did not react with MS-2 cells when they produced regressing tumors. MS-2 cells producing regressing tumors reacted with serum from mice in which the MS-2 tumor had regressed and with serum from mice immunized with MS-2T cells at late tct when they were poorly oncogenic. The antigenic changes seemed, therefore, to parallel the decrease of malignancy. A chromosomal analysis carried out on MS-2 and MS-2T cells, when both produced progressive tumors, showed a modal number of 48 and 44, respectively. MS-2T cells showed a large acrocentric chromosome. In contrast, the MS-2 cells at late tct, when they gave regressing tumors, showed a modal number of 60 and a wide range of distribution of chromosome number.
Asunto(s)
Línea Celular , Virus de la Leucemia Murina de Moloney , Sarcoma Experimental , Animales , Antígenos de Neoplasias , Transformación Celular Neoplásica , Células Cultivadas , Cromosomas/análisis , Ratones , Trasplante de Neoplasias , Sarcoma Experimental/inmunologíaAsunto(s)
Técnicas de Cultivo , ADN/biosíntesis , Daunorrubicina/farmacología , Mitosis/efectos de los fármacos , ARN/biosíntesis , Animales , Autorradiografía , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , ADN/antagonistas & inhibidores , Fibroblastos/metabolismo , Músculos , Nucleósidos/metabolismo , ARN/antagonistas & inhibidores , Ratas , Timidina/metabolismo , Factores de Tiempo , Tritio , Uridina/metabolismoAsunto(s)
Antraquinonas/síntesis química , Antineoplásicos/síntesis química , Daunorrubicina/análogos & derivados , Nucleósidos/síntesis química , Animales , Antraquinonas/farmacología , Antraquinonas/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Arabinosa/síntesis química , Arabinosa/farmacología , Arabinosa/uso terapéutico , Células Cultivadas , Daunorrubicina/síntesis química , Doxorrubicina/análogos & derivados , Embrión de Mamíferos/efectos de los fármacos , Células HeLa/efectos de los fármacos , Técnicas In Vitro , Ratones , Virus de la Leucemia Murina de Moloney/efectos de los fármacos , Nucleósidos/farmacología , Nucleósidos/uso terapéutico , Sarcoma 180/tratamiento farmacológicoAsunto(s)
Antineoplásicos/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Rodaminas/farmacología , Xantenos/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Luz , Neoplasias , Rodamina 123RESUMEN
Doxorubicin-induced DNA damage was studied in the P388 leukemia cell line and in a doxorubicin-resistant subline by alkaline elution techniques. DNA single-strand breaks and DNA-protein cross-links were determined. Whereas, in the sensitive line, 1 hr exposure to drug induced DNA damage in a concentration-dependent manner, in the resistant line only a marginal effect was observed at high drug concentrations. In contrast, elution kinetics of DNAs from cells irradiated with X-rays were similar in both lines. Although a reduced intracellular drug accumulation was found in resistant cells, this difference could not account for the marked reduction in doxorubicin-induced DNA damage. The degree of resistance of the P388 subline was reduced about 7-fold by verapamil, whereas the extent of DNA damage was unaffected. These results suggest the presence of alternative modes of resistance, independent of membrane changes, in highly resistant cells.
Asunto(s)
ADN/análisis , Doxorrubicina/farmacología , Leucemia P388/genética , Leucemia Experimental/genética , Animales , Línea Celular , ADN/efectos de la radiación , Resistencia a Medicamentos , Matemática , Ratones , Factores de Tiempo , Verapamilo/farmacologíaRESUMEN
Fibroblasts and lymphocytes are the most widely used cells for studying the so-called biostimulative effect of low-power laser in vitro. In contrast, stimulation of cancer cells by laser light has not been investigated extensively. The present study attempted to evaluate whether or not human tumor cells could exhibit an increase in colony-forming capability following low-watt laser irradiation. LoVo and HT29 (colon carcinoma), MCF7 (breast carcinoma), M14 and JR1 (malignant melanoma) cell lines were irradiated at different doses of light delivered from an argon or an argon-dye laser. Radiant exposures between 4.2 and 150 kJ/m2 at irradiances ranging from 35 to 500 W/m2 were delivered. Results were mixed. Of the 41 experiments performed, five showed a significant statistical increase in the number of colonies (P less than 0.05), whereas three showed a decrease (P less than 0.05). Nevertheless, the trend of most data was toward an increase in colony formation, and Wilcoxon's signed-ranks test suggested that light increases tumor cell culture growth (P less than 0.03).
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Rayos Láser , Ensayo de Tumor de Célula Madre , Neoplasias de la Mama/patología , División Celular/efectos de la radiación , Neoplasias del Colon/patología , Humanos , Técnicas In Vitro , Melanoma/patología , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
The detection of low amounts of anthracyclines in single cells was attained with a microscope-photometer by employing an argon laser as a fluorescence excitation source. The time course of drug uptake was followed by incubating the cells under the microscope and measuring the increase of fluorescence intensity from the beginning of the drug penetration. An intracellular distribution map of the drug fluorescence was obtained by scanning measurements, through which the more specific sites of binding were visualized. This means of detection is compared with two of the most commonly employed biochemical techniques.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Rayos Láser , Espectrometría de Fluorescencia/métodos , Antineoplásicos/metabolismo , Daunorrubicina/análogos & derivados , Daunorrubicina/metabolismo , Células HeLa/metabolismo , Humanos , Idarrubicina , Cinética , Naftacenos/metabolismoRESUMEN
Daunorubicin was bound to poly-L-aspartic acid via the methylketone side chain of the drug to avoid reaction of the sugar amino group believed to be essential for optimal drug activity. Attachment of the drug to the polyamino acid by an ester linkage was achieved by nucleophylic substitution reaction of 14-bromo-daunorubicin. Compared with free daunorubicin, the polymeric derivative was less cytotoxic to HeLa cells in vitro, but more effective against all tumor models tested (P388 leukemia, Gross leukemia, MS-2 sarcoma). The binding to the polypeptide markedly reduced drug toxicity but only slightly decreased drug potency. The daunorubicin-poly-L-aspartic acid conjugate demonstrated antitumor activity comparable to that of doxorubicin in leukemia models, but superior to that of doxorubicin in a solid tumor model (MS-2 sarcoma).