RESUMEN
To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.
Asunto(s)
Clorofila/análogos & derivados , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/farmacocinética , Animales , Clorofila/química , Clorofila/farmacocinética , Clorofila/farmacología , Humanos , Dosificación Letal Mediana , Ratones , Microscopía Fluorescente , Mutación , Fármacos Fotosensibilizantes/química , Fracciones Subcelulares/metabolismo , Células Tumorales CultivadasRESUMEN
The cationic photosensitizing triaryl methane dye Victoria Blue BO (VBBO) localizes in mitochondria and causes oxidative damage to this organelle during photodynamic therapy (PDT). Oxidative stresses from other photosensitizers induce a variety of stress proteins. The endoplasmic reticulum (ER)-based, calcium-binding stress protein GRP78 is a putative protective factor for photosensitizers such as Photofrin that damage multiple intracellular sites and for several cytotoxic agents. In the current study VBBO-PDT was found to induce glucose-regulated protein (GRP)78. However, in contrast to other drugs, rather than being protected, human squamous carcinoma cells (FaDu) induced to express GRP78 by calcium ionophore A23187 became more sensitive to PDT. A line of Chinese hamster ovary cells (C-1) constitutively overexpressing GRP78 also were more sensitive. Cytotoxicity of the A23187 treatment and VBBO was synergistic, with more than 11-fold potentiation with light irradiation, but was only additive in the dark. The increased cell killing was not due to differences in VBBO uptake or to changes in the intracellular localization of VBBO caused by calcium ionophore or GRP78. Thus, GRP78 appears to enhance rather than protect against VBBO-induced mitochondrial photodamage and contributes to cell death. This novel finding possibly may stem from the effects of GRP78, ER Ca2+ stores and ATP consumption on the Ca2+ and ATP-dependent mitochondrial permeability transition that may be evoked by PDT damage to the mitochondrial respiratory chain. The work suggests interventions that may potentiate PDT with mitochondrial targeting sensitizers and potential enhancements in efficacy when GRP78 is upregulated biologically or pharmacologically.
Asunto(s)
Calcimicina/farmacología , Proteínas Portadoras/biosíntesis , Proteínas de Choque Térmico , Ionóforos/farmacología , Mitocondrias/metabolismo , Chaperonas Moleculares/biosíntesis , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Compuestos de Amonio Cuaternario/farmacología , Animales , Células CHO , Cricetinae , Sinergismo Farmacológico , Chaperón BiP del Retículo Endoplásmico , Humanos , Fármacos Fotosensibilizantes/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Células Tumorales CultivadasRESUMEN
Endogenously generated protoporphyrin IX (PpIX) from exogenous ALA can be an effective photosensitizer. PpIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PpIX to heme. Iron also is necessary for cell replication. Since iron can be toxic, intracellular iron levels are tightly controlled. Activated and proliferating cells respond to the demand for intracellular iron by upregulating membrane expression of the transferrin receptor (CD71) which is needed for iron uptake. We predicted that activated lymphocytes (CD71+) would preferentially accumulate PpIX because of their lower intracellular iron levels and because of competition for iron between ALA-induced heme production and cellular growth processes. Thus, the CD71+ cells could serve as PDT targets. Stimulation of human peripheral blood lymphocytes (PBL) with the mitogens, phytohemagglutinin A, concanavalin A and pokeweed prior to incubation with ALA results in PpIX accumulation correlating with level of activation. Activated lymphocytes expressing high levels of surface CD71 transferrin receptors generated more PpIX than those with low CD71 expression. Incubating activated cells in transferrin depleted medium (thereby decreasing the iron availability) further increased PpIX levels. Malignant, CD71+ T lymphocytes from a patient with cutaneous T-cell lymphoma (CTCL)/Sezary syndrome also accumulated increased PpIX levels in comparison to normal lymphocytes. PDT of activated lymphocytes and Sezary cells after ALA incubation demonstrated preferential killing compared to normal, unstimulated PBL. These findings suggest a possible mechanism for the selectivity of ALA PDT for activated CD71+ cells. They also indicate a clinical use for ALA-PDT in therapy directed towards the malignant lymphocytes in leukemias and lymphomas, and as animmunomodulatory agent.
Asunto(s)
Ácido Aminolevulínico/farmacología , Activación de Linfocitos , Linfocitos/fisiología , Fotoquimioterapia , Protoporfirinas/metabolismo , Receptores de Transferrina/fisiología , Síndrome de Sézary/fisiopatología , Neoplasias Cutáneas/fisiopatología , Células Cultivadas , Hemo/metabolismo , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Mitógenos , Protoporfirinas/análisis , Receptores de Transferrina/análisis , Valores de Referencia , Síndrome de Sézary/inmunología , Neoplasias Cutáneas/inmunologíaRESUMEN
A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 M-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.
Asunto(s)
Marcadores de Afinidad/química , Colorantes Fluorescentes/química , Xantenos/química , Marcadores de Afinidad/síntesis química , Estabilidad de Medicamentos , Citometría de Flujo , Colorantes Fluorescentes/síntesis química , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Microscopía Fluorescente , Estructura Molecular , Espectrometría de Fluorescencia , Espectrofotometría , Relación Estructura-Actividad , Xantenos/síntesis químicaRESUMEN
Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei.
Asunto(s)
Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/química , Compuestos Organometálicos/química , Compuestos Organofosforados/química , Línea Celular , Dextranos/química , Células Epiteliales , Estudios de Evaluación como Asunto , Humanos , Inmunoglobulina G/química , Lectinas/química , Proteínas de la Membrana/química , Microscopía Fluorescente , Sensibilidad y Especificidad , Espectrometría de FluorescenciaRESUMEN
A series of fluorescent, long-wavelength, benzo[c]-xanthene dyes has been characterized for pH measurement in both excitation and emission ratio applications. The two general classes of these indicators are seminaphthofluoresceins (SNAFLs) and seminaphthorhodafluors (SNARFs) which are substituted at the 10-position with oxygen or nitrogen, respectively. These probes show separate emissions from the protonated and deprotonated forms of the fluorophores. The dyes may be excited at 488 or 514 nm with argon ion lasers. Most of the indicators have pKa values between 7.6 and 7.9. Detailed photophysical studies were conducted on the carboxy-SNAFL-1 system and excited-state prototropic reactions were compared to structurally related derivatives, such as the umbelliferones. Membrane permeant esters, such as diacetates and acetoxymethyl esters have also been prepared. The indicators are spectrally well resolved from calcium indicators such as fura-2 and indo-1 and should be suitable for simultaneous determination of pH and Ca2+ transients.
Asunto(s)
Colorantes Fluorescentes/química , Indicadores y Reactivos , Xantenos/química , Benzopiranos , Concentración de Iones de Hidrógeno , Estructura Molecular , Naftoles/química , Análisis de Regresión , Espectrometría de Fluorescencia , Análisis Espectral , TemperaturaRESUMEN
Dihydrotetramethylrosamine, a fluorogenic substrate for peroxidase, and its fluorescent oxidation product, tetramethylrosamine chloride, were evaluated. The substrate is colorless and nonfluorescent while the oxidized dye absorbs at 550 nm and emits at 574 nm in both methanol and water. In vitro assays demonstrated that the substrate was oxidized to the fluorophore by horseradish peroxidase in the presence of hydrogen peroxide. In vivo uptake and oxidation of the substrate by Amoeba proteus was characterized by the initial appearance of fluorescent phagocytic vacuoles with subsequent localization in vesicular organelles the size and shape of protozoan mitochondria. Similar staining patterns occurred in cells incubated with substrate, oxidized rosamine or rhodamine 123, a known mitochondrial stain.
Asunto(s)
Colorantes Fluorescentes , Peroxidasas/metabolismo , Fagocitos/metabolismo , Rodaminas , Amoeba/metabolismo , Animales , Microscopía Fluorescente , Oxidación-Reducción , Rodaminas/metabolismo , Espectrometría de Fluorescencia , Análisis EspectralRESUMEN
Addition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF-AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, 1) a fast alkalinization, reaching a maximum pH in approximately 2-5 min; 2) a slow alkalinization, reaching a maximum pH in 10-20 min; 3) a very slow alkalinization, not reaching a plateau pH within the measurement time; 4) no apparent change in pH during the measurement time; 5) an early transient acidification, followed by either a fast or slow alkalinization; and 6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of approximately 0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.
Asunto(s)
Citoplasma/fisiología , Fibroblastos/efectos de los fármacos , Animales , Medios de Cultivo/farmacología , Citoplasma/análisis , Dextranos , Fibroblastos/fisiología , Fluoresceínas , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos , Microscopía ElectrónicaRESUMEN
A simple, rapid colorimetric method is described which determines procainamide in commercially available, injectable solutions. It is based on complexing the drug with cupric ion and then measuring the absorption at 380 nm. The reaction is fast and the metal-drug ratio in the complex is 1:1. The pH optimum for maximum reaction is 4.0-4.5 which is maintained by an acetate buffer. Linear standard curves were obtained from 1.52 to 6.06 mg/mL. Analysis of 29 commercial samples resulted in an average of 97.3% of the label claim.