RESUMEN
We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 microM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Liposomas/química , Neoplasias Pulmonares/tratamiento farmacológico , Antineoplásicos/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cisplatino/farmacocinética , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologíaRESUMEN
Fluorescence method was used to study the interactions of anthracyclines with purified DNA and with cell nuclei at 37 degrees C, at pH ranging from 6.8 to 8. Four anthracyclines were used; adriamycin (ADR), 4'-o-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR) and aclacinomycin (ACM). The values of pKa of deprotonation of these four drugs in the pH range 6.5-8.5 are 8.4, 7.7, 8.4 and 7.0 for ADR, THP-ADR, DNR and ACM, respectively. The overall binding constants K* of these four drugs to purified DNA was determined at various pH values. The binding constants K0 and K+ of the respectively neutral form and once protonated form of the drugs to DNA were calculated. Using cell nuclei from K562 cells, the amount of drug intercalated (CN) within the nuclei of K562 cells and the amount of free drug (CE) in the solution were determined at various pH values: measuring at the same pH values, a linear correlation occurred between K* and CN/CE.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Núcleo Celular/metabolismo , ADN/metabolismo , Aclarubicina/análogos & derivados , Aclarubicina/metabolismo , Antineoplásicos/metabolismo , Daunorrubicina/metabolismo , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Cinética , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The interaction of mitoxantrone, ametantrone and their Pd(II) complexes with DNA have been studied using absorption and circular dichroism spectroscopy. We have shown that mitoxantrone forms with Pd(II) a complex in which two Pd(II) ions are bound to two molecules of drug (D1 and D2). One Pd(II) ion is bound to the two nitrogens of the side chain on C-5 of molecule D1 and to the two nitrogens of the side chain on C-5 of molecule D2, whereas the second Pd(II) ion is bound to the nitrogens of the side chain on C-8 of molecule D1 and of molecule D2. The same complex is formed between Pd(II) and ametantrone. The stability constants for these complexes are, respectively, beta M = (1.4 +/- 0.5).10(19) and beta A = (2.5 +/- 0.5).10(18). They display antitumor activity against P 388 leukemia which compares with that of the free drugs. Interactions of the free drugs with DNA have been studied. Mitoxantrone and ametantrone are not optically active by themselves. However, through interaction with DNA, there is an induction of optical activity within the electronic transitions of both drugs. At a nucleotide/drug molar ratio lower than about 5 a CD signal of the couplet type is observed, suggesting that there is a coupling between the pi----pi transitions of the molecules of drugs intercalated between the base pairs. This coupling disappears when the molar ratio is increased. The interactions of the Pd(II) complexes with DNA do not give rise to induction of optical activity within the electronic transition of the drugs, indicating that the presence of the metal ion prevents the intercalation of the drugs between the base pairs.
Asunto(s)
ADN/metabolismo , Mitoxantrona/análogos & derivados , Mitoxantrona/metabolismo , Paladio/metabolismo , Animales , Dicroismo Circular , Leucemia P388/metabolismo , Modelos Químicos , EspectrofotometríaRESUMEN
In the presence of cardiolipin-containing small unilamellar vesicles, the antitumor compound adriamycin loses its ability to catalyse the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. The data strongly suggest that in the presence of cardiolipin the dihydroanthraquinone moiety is embedded in the phospholipid bilayer and thus inaccessible to the enzyme.
Asunto(s)
Antraquinonas/metabolismo , Cardiolipinas/farmacología , Doxorrubicina/metabolismo , Liposomas , Doxorrubicina/farmacología , Transporte de Electrón/efectos de los fármacos , Cinética , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
Fe(III) complex of an antitumoral antibiotic carminomycin has been studied. Using potentiometric and spectroscopic measurements we have shown that carminomycin forms with Fe(III) a well-defined species in which three molecules of drug are chelated to one Fe(III) ion. This occurs with the release of one proton per molecule of drug. Magnetic susceptibility measurements suggest that six oxygen atoms are bound to iron. The stability constant is 3 X 10(34). The in vitro inhibition of P 388 leukemia cell growth by this complex compares with that of the free drug. This complex, unlike the free drug, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase.
Asunto(s)
Carubicina , Reductasas del Citocromo/metabolismo , Daunorrubicina , Hierro , NADH Deshidrogenasa/metabolismo , Compuestos Organometálicos , Superóxidos/metabolismo , Animales , Carubicina/farmacología , División Celular/efectos de los fármacos , Dicroismo Circular , Grupo Citocromo c/metabolismo , Daunorrubicina/análogos & derivados , Estabilidad de Medicamentos , Caballos , Concentración de Iones de Hidrógeno , Hierro/farmacología , Leucemia P388/patología , Ratones , Miocardio , Potenciometría , EspectrofotometríaRESUMEN
The interaction of mithramycin (MTR) with Ca2+, Cd2+, Tb3+, Gd3+, Li+, Na+ and K+ ions has been studied by circular dichroism and absorption spectroscopy. Mithramycin binds strongly to Ca2+, Cd2+, Tb3+ and Gd3+ forming a 1:4 Ca2+: MTR entities with a left-handed screw conformation. The concentration of Ca2+ present in water currently used being about 10 microM, this leads to the conclusion that, in most of the experiments reported in the literature, about 40 microM mithramycin were actually bound to Ca2+. Mithramycin also binds to Na+, forming entities with left-handed screw conformation, but not to K+ and Li+. None of these cations were able to promote the mithramycin-DNA interaction.
Asunto(s)
Quelantes/metabolismo , Metales/metabolismo , Plicamicina/metabolismo , Animales , Secuencia de Carbohidratos , Cationes/metabolismo , Bovinos , Dicroismo Circular , ADN/metabolismo , Ácido Egtácico/química , Datos de Secuencia MolecularRESUMEN
5-Iminodaunorubicin is an anthracycline derivative exhibiting promising antitumor activity. Using potentiometric and spectroscopic measurements we have shown that 5-iminodaunorubicin forms with Fe(III) a complex in which three molecules of drug are bound to one Fe(III) ion. Each molecule is chelated through the C-12-carbonyl and the C-11-phenolate oxygen atoms. The stability constant is 1.6 X 10(34). Using circular dichroism measurements we have studied the interactions of 5-iminodaunorubicin with cardiolipin-containing vesicles. We have shown that cardiolipin could bind one molecule of drug without penetration of the dihydroanthraquinone moiety into the bilayer.
Asunto(s)
Cardiolipinas , Daunorrubicina/análogos & derivados , Compuestos Férricos , Hierro , Membrana Dobles de Lípidos , Dicroismo Circular , Concentración de Iones de Hidrógeno , Potenciometría , Análisis EspectralRESUMEN
The interaction of the antitumor compound adriamycin with human erythrocyte membranes, used as models of target cell membranes, has been studied using circular dichroism measurements. In order to elucidate the nature of the sites involved in the electrostatic interaction between adriamycin and erythrocyte membranes, its interaction with the following macromolecular systems was studied: phosphatidylserine-containing small unilamellar vesicles (SUV), prepared from total lipid extracts of erythrocytes, sialic acid-depleted erythrocyte ghosts and mucopolysaccharides. We have shown that the interaction between adriamycin and carboxylate groups is very weak and that negatively charged phosphate groups, in the case of membranes, or sulfate groups, in the case of mucopolysaccharides, are responsible for the prime interaction of adriamycin with these macromolecular systems.
Asunto(s)
Doxorrubicina/sangre , Membrana Eritrocítica/metabolismo , Fosfolípidos/sangre , Dicroismo Circular , Electroquímica , Glicosaminoglicanos/sangre , Humanos , Liposomas/metabolismo , Sustancias Macromoleculares , Lípidos de la Membrana/sangre , Ácido N-Acetilneuramínico , Neuraminidasa/farmacología , Fosfatidilcolinas/sangre , Fosfatidilserinas/sangre , Pronasa/farmacología , Ácidos Siálicos/sangre , Espectrina/fisiología , Tripsina/farmacologíaRESUMEN
A fluorescence method was used to follow the interaction of 4'-o-tetrahydropyranyladriamycin (THP-ADR) with drug-resistant and -sensitive K562 cells. The amounts of drug bound to the nuclei at the steady state, Cn and at the equilibrium state, CN, once the membrane has been solubilized with Triton X-100, have been determined as a function of the pH outside the cells (pHe): Cn increased and CN decreased as pHe increased. At a given pH value outside the cells, CN is the same for both sensitive and resistant cells, whereas Cn is lower in resistant cells as compared to sensitive cells. Using the observation that the essential binding characteristics of THP-ADR in nuclei are the same for both types of cell, the osmotic active drug concentration, Ci, in the cytoplasm of the cells was determined at different values of pHe. Using fluorescent dye, the cytoplasmic pH was determined and found equal to 7.2 +/- 0.1 in both types of cell. In sensitive cells, the equilibrium transmembrane concentrations verified the relation [DH+]i/[DH+]e = [H +]i/[H+]e where [DH +]i and [DH +]e stand for the concentration of protonated form of the drug inside and outside the cells, respectively. This indicates that the uptake of the drug occurs through free permeation of the neutral form of the drug in response to delta pH gradient. Such a relation is not verified in the case of resistant cells.
Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , Citoplasma/metabolismo , Resistencia a Medicamentos/fisiología , Línea Celular , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Ósmosis , Espectrometría de FluorescenciaRESUMEN
The uptake of three anthracycline derivatives: doxorubicin, daunorubicin and pirarubicin, into large unilamellar vesicles (LUV) in response to a driving force provided by DNA encapsulated inside the LUV has been investigated as a function of the temperature and of the bilayers lipid composition. The kinetics of the decay of the anthracycline fluorescence in the presence of DNA-containing liposome was used to follow the diffusion of the drug through the membrane. For the three drugs, the permeability coefficient of the neutral form of the drug (P0) decreases as the amount of negatively charged phospholipid in the bilayers increases. This can be explained by the fact that the kinetics of passive diffusion of the drugs depends on the amount of neutral form embedded in the polar head group region, which decreases as the quantity of negatively charged phospholipids increases. P0 also decreases as the amount of cholesterol, that makes the bilayer more rigid, increases. The activation energies, Ea, for the passage of the neutral form of these anthracyclines through the bilayers lie within 100 +/- 15 kJ x ml-1, except for pirarubicin and doxorubicin through anionic phospholipid-rich membranes (Ea = 57 kJ x mol-1) and cholesterol-rich membranes (Ea = 167 kJ x mol-1).
Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , ADN/análisis , Membrana Dobles de Lípidos/metabolismo , Colesterol/farmacología , Daunorrubicina/farmacocinética , Difusión , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Composición de Medicamentos , Fluorescencia , Cinética , Membrana Dobles de Lípidos/química , Liposomas/química , Liposomas/metabolismo , Estructura Molecular , Permeabilidad , Fosfolípidos/química , Conformación Proteica , Temperatura , TermodinámicaRESUMEN
Doxorubicin, daunorubicin and other anthracycline antibiotics constitute one of the most important groups of drugs used today in cancer chemotherapy. The details of the drug interactions with membranes are of particular importance in the understanding of their kinetics of passive diffusion through the membrane which is itself basic in the context of multidrug resistance (MDR) of cancer cells. Anthracyclines are amphiphilic molecules possessing dihydroxyanthraquinone ring system which is neutral under the physiological conditions. Their lipophilicity depends on the substituents. The amino sugar moiety bears the positive electrostatic charge localised at the protonated amino nitrogen. The four anthracyclines used in this study doxorubicin, daunorubicin, idarubicin and idarubicinol (an idarubicin metabolite readily formed inside the cells) have the same amino sugar moiety, daunosamine, with pKa of 8.4. Thus, all drugs studied will exhibit very similar electrostatic interactions with membranes, while the major differences in overall drug-membrane behaviour will result from their hydrophobic features. Circular dichroism (CD) spectroscopy was used to understand more precisely the conformational aspects of the drug-membrane systems. Large unilamellar vesicles (LUV) consisting of phosphatidylcholine, phosphatidic acid (PA) and cholesterol, were used. The anthracycline-LUV interactions depend on the molar ratio of phospholipids per drug. At low molar ratios drug:PA, these interactions depend also on the anthracycline lipophilicity. Thus, both doxorubicin and daunorubicin bind to membranes as monomers and their CD signal in the visible is positive. However, doxorubicin with its very low lipophilicity binds to the LUV through electrostatic interactions, with the dihydroxyanthraquinone moiety being in the aqueous phase, while daunorubicin, which is more lipophilic is unable to bind only through electrostatic interactions and actually the hydrophobic interactions are the only detected. The highly hydrophobic idarubicin, forms within the bilayer a rather complex entity involving 2-3 molecules of idarubicin associated in the right-handed conformation, one cholesterol molecule and also molecule(s) of phosphatidic acid, as this special oligomeric species is not detected in the absence of negatively-charged phospholipids. Idarubicinol differs from idarubicin with CH(13)-OH instead of C(13)=O and its interactions with LUV are distinctly different. Its CD signal in the visible becomes negative and no self associations of the molecule within the bilayer could be detected. The variation of the sign of the Cotton effect (positive to negative) may derive from the changes in the C(6a)-C(7)-O(7)-C(1') dihedral angle. It is noteworthy that C(13)-OH group, which strongly favours formation of the dimeric species in aqueous solutions when compared to idarubicin prevent association inside the LUV bilayer. At high ratios of phospholipids per drug all of them are embedded within the bilayer as monomer.
Asunto(s)
Antraciclinas/química , Membrana Dobles de Lípidos/química , Liposomas/química , Dicroismo Circular , Daunorrubicina/análogos & derivados , Daunorrubicina/química , Dimerización , Doxorrubicina/química , Idarrubicina/química , Membranas Artificiales , Modelos Moleculares , SolventesRESUMEN
The decrease of the intracellular concentration of drug in resistant cells as compared to sensitive cells is, in most of cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein (P-gp) responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to follow the P-gp-associated efflux of these drugs in the absence or presence of verapamil. In the present study, 4'-o-tetrahydro-pyranyladriamycin (THP-adriamycin) was used. Two different methods were used to determine the kinetics of active efflux of THP-adriamycin: (1) at the steady-state, (2) directly, after the addition of glucose to cells first incubated with THP-adriamycin in the presence of N3- and in the absence of glucose. Kinetic analysis indicates: (1) a saturation of the active efflux when the cytosolic free drug concentration increased (the Michaelis constant Km = 0.5 +/- 0.3 microM) and (2) that the inhibitory effect of verapamil on P-gp-associated efflux of THP-adriamycin in living cells is non-competitive.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Doxorrubicina/análogos & derivados , Glicoproteínas de Membrana/antagonistas & inhibidores , Verapamilo/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Sitios de Unión , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Resistencia a Medicamentos , Humanos , Cinética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been studied. The kinetics of uptake and the nuclear concentration of anthracyclines in human lymphocytes have thus been determined using the fluorescence properties of these drugs. Four anthracyclines have been used: adriamycin (ADR), 4'-O-tetrahydropyranyl-adriamycin (THP-ADR), carminomycin (CAR) and aclacinomycin A (ACM). We have shown that no quenching of the drug fluorescence is obtained through interaction of the drugs with the various components of the cell except the nucleus. No quenching is observed with cells lacking nucleus such as platelets and erythrocytes. Quenching of drug fluorescence occurs when drugs intercalate between base pairs of DNA only. This allows rapid determination of the amount of drug intercalated between nuclear base pairs in the steady state. We have thus estimated that one molecule of drug can bind for every 10, 8.3, 10 and 6.7 DNA base pairs in the case of ADR, THP-ADR, ACM and CAR, respectively. The kinetics of drug incorporation into the nucleus, once the cells have been solubilized with triton X-100, is very fast for the four drugs. However, the kinetics of drug uptake by the intacts cells strongly depends on the nature of the drug. When 10(9) cells/l are incubated with 1 microM drug, 50% of the final nuclear concentration is reached within 97 min, 3.2 min, 3.0 min and 1.2 min in the case of ADR, THP-ADR, CAR and ACM, respectively. The kinetics directly correlates with the polarity of the molecule.
Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , Núcleo Celular/análisis , Linfocitos/metabolismo , Aclarubicina/análogos & derivados , Aclarubicina/farmacocinética , Antibióticos Antineoplásicos/análisis , Composición de Base , Sitios de Unión , Plaquetas/análisis , Carubicina/farmacocinética , ADN/metabolismo , Doxorrubicina/farmacocinética , Eritrocitos/análisis , Humanos , Estructura Molecular , Espectrometría de FluorescenciaRESUMEN
The interaction of phorbol 12,13-dibutyrate (PDBu), 12-O-retinoylphorbol 13-acetate (RPA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) with L-alpha-phosphatidylserine-containing small unilamellar vesicles or erythrocyte ghosts was monitored by circular dichroism (CD). No change in the CD spectra of PDBu was observed upon binding, while RPA and TPA spectra were slowly affected by the interaction. The changes in RPA and TPA spectra were assigned to the embedding of these molecules in the membrane bilayers. In the presence of 10(8) cells/ml, after one minute incubation, about 2 to 5% of the amount of phorbol ester added is embedded in the membrane. It is suggested that either phorbol esters entering the membrane is not a prerequisite for protein kinase C activation or the amount of phorbol esters necessary to activate protein kinase C is very small.
Asunto(s)
Dicroismo Circular , Membrana Eritrocítica/metabolismo , Liposomas/metabolismo , Ésteres del Forbol/metabolismo , Análisis Espectral , Activación Enzimática , Humanos , Forbol 12,13-Dibutirato/metabolismo , Fosfatidilserinas/metabolismo , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/metabolismoRESUMEN
Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins, the 170-kDa P-glycoprotein (Pgp) and the 190-kDa multidrug resistance-associated protein (MRP1). One possible pharmacological approach to overcome drug resistance is the use of specific inhibitors, which enhance the cytotoxicity of known antineoplastic agents. However, while many compounds have been proven to be very efficient in inhibiting Pgp activity only some of them are able to inhibit MRP1. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs with physicochemical properties favoring the uptake of the drug by the resistant cells. The intracellular drug retention influences its cytotoxic effect. The level of the intracellular drug content is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). In this work, the kinetics of drug uptake and the kinetics of active efflux of several anthracycline derivatives in both Pgp expressing K562/Adr cells and MRP1 expressing GLC4/Adr cells was determined. Our data have shown that in both cell lines there is no correlation between the resistance factor and the kinetics of drug efflux by these pumping systems. However, a very good correlation between the resistance factor and the kinetics of drug uptake has been established in both cell lines: the resistance factor decreases when the kinetics of drug uptake increases. This work has clearly shown that when the rate of transmembrane transport of anthracycline is high enough, the efflux mediated by the protein transporter is not able to pace with it. The protein transporter essentially operates in a futile cycle and the resistance factor is tending to one. It does not mean, however, that when the resistance factor is close to one the anthracycline is not transported by the pump.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antraciclinas/metabolismo , Membrana Celular/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Antraciclinas/química , Antraciclinas/farmacología , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Daunorrubicina/farmacología , Resistencia a Medicamentos , Humanos , Cinética , Estructura Molecular , Proteína 3 Homóloga de MutS , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed. The three daunorubicin molecules that bind to one Fe(III) are not chemically modified through complexation with iron. In the case of the Fe(III)-adriamycin and Fe(III)-4'-O-tetrahydropyranyladriamycin complexes, about one of the three anthracycline molecules is chemically modified, yielding a highly lipophilic derivative, the 7,8-dehydro-9,10-desacetyladriamycin. The others molecules remain unchanged, i.e., highly hydrophilic in the case of adriamycin. These two species have a different fluorescent spectrum and can be identified inside the cell, using microspectrofluorometry. In the case of the Fe(III)-adriamycin complex, the lipophilic derivative is more rapidly internalized in the cell than the hydrophilic one. Diffusion into the plasmic membrane is the limiting step for the uptake of anthracycline by cells; this means that the plasmic membrane speeds up the dissociation of the Fe(III)-anthracycline complex.
Asunto(s)
Membrana Celular/metabolismo , Daunorrubicina/análogos & derivados , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Hierro/metabolismo , Compuestos Organometálicos/metabolismo , Animales , Línea Celular , Daunorrubicina/química , Difusión , Doxorrubicina/química , Hierro/química , Compuestos Organometálicos/química , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.
Asunto(s)
Resistencia a Múltiples Medicamentos , Leucemia/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Citometría de Flujo , Humanos , Inmunofenotipificación , Fenotipo , Células Tumorales CultivadasRESUMEN
Hydroxyrubin (OH-Dox), a neutral doxorubicin derivative that is only slightly cross-resistant to doxorubicin (Dox), can be actively pumped out of resistant K562 cells by P-glycoprotein (P-gp). This efflux is saturable and can be inhibited by verapamil. The Michaelis constant is equal to 2 +/- 0.5 microM. However, the efficiency of P-gp in pumping out the drugs is 2.5 times less for OH-Dox than for Dox. This shows that in order to be pumped out by P-gp a molecule does not necessarily have to have a basic center. The mean influx coefficient for the drug is 5 times higher for OH-Dox than for Dox. In conclusion, the degree of resistance of analogs is related not only to their ability to be recognized and transported by P-gp but also, and probably essentially, to their kinetics of uptake. Both parameters have to be taken into account in the rational design of new compounds capable of overcoming multidrug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Medicamentos , Epirrubicina/análogos & derivados , Línea Celular , Células Clonales , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Epirrubicina/metabolismo , Epirrubicina/farmacocinética , Glucosa/farmacología , Humanos , Cinética , Leucemia Eritroblástica Aguda , Estructura Molecular , Células Tumorales CultivadasRESUMEN
Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins--the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1)--that pump drugs out of MDR cells. The intracellular level of a drug, which influences the drug's cytotoxic effect, is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). One possible pharmacological approach to overcoming drug resistance is the use of specific inhibitors that enhance the cytotoxicity of known antineoplastic agents. Many compounds have been proven to be very efficient in inhibiting P-gp activity, but only some of them can inhibit MRP1. However, the clinical results obtained so far by this approach have been rather disappointing. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs whose physicochemical properties favor the uptake of such drug by resistant cells. Our recent studies have shown that whereas the P-gp- and MRP1-mediated efflux of different anthracycline-based drugs may not differ considerably, their kinetics of uptake do. Thus, the high uptake of drug by cells may lead to concentrations at the cellular target site high enough to achieve the needed cytotoxicity against MDR cells. Therefore, increased drug lipophilicity might be one factor in improving drug cytotoxicity in MDR cells. In vitro studies have shown that idarubicin, an analogue of daunorub cin, is more effective than daunorubicin and doxorubicin against MDR tumor cell lines and that this increased effectiveness is related in part to the increased lipophilicity of idarubicin. Other studies have also confirmed the strong impact of lipophilicity on the uptake and retention of anthracyclines in MDR cells.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacología , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/genética , Humanos , Células Tumorales CultivadasRESUMEN
We demonstrated the existence of three transport activities in promastigotes of Leishmania braziliensis, Leishmania guyanensis, and Leishmania mexicana. The first activity, an energy-dependent efflux of pirarubicin, was observed in all Leishmania species and inhibited by verapamil, by 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-py ridinecarboxylate P oxide (PAK104P) and by the phenothiazine derivatives: thioridazine, prochlorperazine, trifluoperazine, chlorpromazine and trifluoropromazine. The second activity, an energy-dependent efflux of calcein acetoxymethylester, was observed in all Leishmania species and inhibited by PAK104P and the same phenothiazine derivatives, but not by verapamil. The third activity, an energy-dependent efflux of calcein, was clearly detected in L. braziliensis and guyanensis and inhibited only by prochlorperazine and trifluoperazine. The fact that prochlorperazine and trifluoperazine inhibited the energy-dependent efflux of the three substrates suggests that these activities are mediated by the same transport system. It is noteworthy that the transport system identified in this study shares several properties with the mammalian multidrug resistance pump, MRP1. Pirarubicin, calcein acetoxymethylester and calcein are well known substrates of the MRP. Furthermore, the three types of inhibitors are also inhibitors of the MRP function.