RESUMO
Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry.
Assuntos
Benzimidazóis/metabolismo , Plaquetas/metabolismo , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose/fisiologia , Preservação de Sangue/métodos , Cães , Citometria de Fluxo/métodos , Potencial da Membrana Mitocondrial/fisiologiaRESUMO
Cy3 and Cy5 dyes linked to the 5' end of a double stranded DNA molecule are known to attach to both strands in a way that is very similar to an additional base pair and has a stabilizing effect on the oligonucleotide. Here we adapt the Peyrard-Bishop mesoscopic model to incorporate cyanine dyes and use the technique of thermal equivalence to obtain the appropriate parameters from existing melting temperatures. We have found that the stacking parameters are in the same range of ordinary AT and CG base pairs, in particular Cy3-A was found to be most rigidly stacked. While the cyanines stabilize the AT hydrogen bonds quite strongly the CG bonds are mostly unaffected.
Assuntos
Carbocianinas/química , DNA/química , Pareamento de Bases , Carbocianinas/metabolismo , DNA/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Desnaturação de Ácido Nucleico , TemperaturaRESUMO
Fluorescent permeant charged probes are commonly used for monitoring the trans-membrane potential in lipid vesicles and biological membranes, which has been earlier described by various mathematical models. In the present study, we developed a more complex model based on the computational step-by-step analysis of the influence of various factors, such as the membrane surface potential, ionic strength, and the aggregation properties of cationic cyanine probe DiSC3(5) in the membrane and aqueous phases, in addition to the Nernstian distribution of the probe across the membrane and the hydrophobic interaction with the lipid bilayer. The final full model allows prediction of the optimal experimental conditions for monitoring the trans-membrane potential, such as the probe/lipid ratio and the concentration of liposomes, with a given percentage of negatively charged phospholipids in the membrane, the ionic strength of the aqueous media, the "membrane-water" partition coefficient and the aggregation properties of the probe, as well as the most adequate mode of fluorescence measurement. In agreement with many experimental studies, this model showed high voltage sensitivity of the quantity of the aqueous phase DiSC3(5) monomers, showing its almost exponential decrease with an increase in the trans-membrane potential value. The model also demonstrated the highest voltage sensitivity of the ratio of the quantity of DiSC3(5) monomers in the aqueous phases to that in the membrane phase. A new combined parameter, the logarithmic function of this ratio, demonstrated almost linear changes within a wide range of the trans-membrane potential changes.
Assuntos
Benzotiazóis/metabolismo , Carbocianinas/metabolismo , Simulação por Computador , Corantes Fluorescentes/metabolismo , Potenciais da Membrana , Benzotiazóis/química , Carbocianinas/química , Membrana Celular/metabolismo , Dimerização , Corantes Fluorescentes/química , Lipossomos/metabolismoRESUMO
BACKGROUND: Due to high optical absorption, triplet quantum yield and affinity to biological structures bichromophoric cyanine dyes (BCDs) can be considered promising sensitizers for application in photodynamic therapy (PDT). In this work, we report on the study of the BCD photocytotoxicity toward melanoma and normal cells in comparison with that of commercial photosensitizer Photogem®. METHODS: The cytotoxic and phototoxic effects were measured by standard tests of cell viability. The drug uptake was obtained by the flow cytometry and optical absorption techniques. The BCD intracellular distribution was obtained by the fluorescence image microscopy using specific organelle markers. RESULTS: Both drugs demonstrated increased cytotoxicity under irradiation, while in darkness their cytotoxic effect at concentrations lower than 20 µM after 24 h of incubation did not exceed 20%. For 5 h of incubation, BCD photocytotoxicity in relation to melanoma cells reached 100% already at concentrations below 5 µM, while for normal cells the effect did not exceed 70% even for the 20 µM concentration. It is shown that BCD penetrates into the cells and is located predominantly in perinuclear cytoplasmic structures. CONCLUSIONS: The BCD photosensitizing characteristics appear more adequate for application in PDT than that of the actually applied commercial photosensitizer Photogem®. Higher light absorption by BCD in the near IR region and its preferential localization in mitochondria can explain its high photocytotoxicity. GENERAL SIGNIFICANCE: BCD can be considered as a new promising photosensitizer class for cancer PDT.
Assuntos
Carbocianinas/farmacologia , Corantes Fluorescentes/farmacologia , Hematoporfirinas/farmacologia , Melanoma Experimental/patologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Animais , Carbocianinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Hematoporfirinas/metabolismo , Humanos , Concentração Inibidora 50 , Melanoma Experimental/metabolismo , Camundongos , Permeabilidade , Fármacos Fotossensibilizantes/metabolismo , Fatores de TempoRESUMO
Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Expressão Gênica , Hepacivirus/fisiologia , Proteínas Proto-Oncogênicas c-bcr/biossíntese , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Subfamília B de Transportador de Cassetes de Ligação de ATP , Carbocianinas/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Hepatócitos/virologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Rodamina 123/metabolismoRESUMO
Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Polímeros/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Etilenodiaminas/química , Humanos , Peso Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Polietilenoglicóis/química , Polímeros/química , Propídio/metabolismo , Propilenoglicóis/química , Rodamina 123/metabolismo , Vimblastina/metabolismoRESUMO
Merozoite release from infected erythrocytes is a complex process, which is still not fully understood. Such process was characterised at ultra-structural level in this work by labelling erythrocyte membrane with a fluorescent lipid probe and subsequent photo-conversion into an electron-dense precipitate. A lipophilic DiIC(16) probe was inserted into the infected erythrocyte surface and the transport of this phospholipid analogue through the erythrocyte membrane was followed up during 48 h of the asexual erythrocyte cycle. The lipid probe was transferred from infected erythrocyte membranes to Maurer's clefts during merozoite release, thereby indicating that these membranes remained inside host cells after parasite release. Fluorescent structures were never observed inside infected erythrocytes preceding merozoite exit and merozoites released from infected erythrocyte were not fluorescent. However, specific precipitated material was localised bordering the parasitophorous vacuole membrane and tubovesicular membranes when labelled non-infected erythrocytes were invaded by merozoites. It was revealed that lipids were interchangeable from one membrane to another, passing from infected erythrocyte membrane to Maurer's clefts inside the erythrocyte ghost, even after merozoite release. Maurer's clefts became photo-converted following merozoite release, suggesting that these structures were in close contact with infected erythrocyte membrane during merozoite exit and possibly played some role in malarial parasite exit from the host cell.
Assuntos
Carbocianinas/metabolismo , Membrana Celular/metabolismo , Eritrócitos/parasitologia , Merozoítos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade , Antígenos de Protozoários , Eritrócitos/química , Merozoítos/química , Plasmodium falciparum/químicaRESUMO
When cultured cells are treated with fluorescent organelle probes or photosensitizer agents, a characteristic redistribution of fluorescence in cell structures occurs frequently after light irradiation. It is currently assumed that such changes, referred to as relocalizations of the fluorescent compounds, represent an important aspect of the photodynamic process, which is based on the excitation of photosensitizers by light in the presence of oxygen. As cell damage and death result from the oxidative stress induced by photodynamic treatments, we have studied here the redistribution of acridine orange (AO) and 3,3'-dimethyl-oxacarbocyanine (DiOC(1)(3)) fluorescence after incubation of HeLa cell cultures with these compounds followed by blue light irradiation to achieve lethal effects. The relocalization of dyes from their original labeling sites (AO: lysosomes, DiOC(1)(3): mitochondria) to nucleic acid-containing structures (cytoplasm, nuclei and nucleoli) appeared clearly associated with cell death. Therefore, the relocalization phenomenon simply reflects fluorescence changes due to the different affinity of these dyes for living and damaged or dead cells. As fluorescent probes are often photosensitizers, prolonged light exposures using fluorescence microscopy will produce lethal photodynamic effects with relocalization of the fluorescent signal and changes in the cell morphology.
Assuntos
Laranja de Acridina/metabolismo , Corantes Fluorescentes/metabolismo , Carbocianinas/metabolismo , Morte Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Mitocôndrias/metabolismoRESUMO
The expression and activity of P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) were analyzed in 178 leukemia samples. Rhodamine-123 (Rho-123) and DiOC(2) were used as substrate to evaluate efflux pump activity. Chronic myeloid leukemia (CML) exhibited a higher percentage of positivity using Rho-123 than DiOC(2) (p=0.000) as compared to other types of leukemia. Moreover, Rho-123 was able to detected Pgp positive cells in a higher proportion of samples than DiOC(2) samples (p=0.004). Similarly, MRP1 positive cells were best detected by Rho-123 as opposed to DiOC(2) (p=0.003). The co-functionality of Rho-123 and DiOC(2) was observed in 26 out of 105 (24.8%) leukemic samples. Co-expression between Pgp and MRP1 was detected in 30 out of 56 (53.6%) samples. As a whole, when the same samples were analyzed, Rho-123 was able to detect Pgp positive cells in a higher proportion of samples than DiOC(2) (p=0.000). Similarly, MRP1 positive cells were best detected by Rho-123 as opposed to DiOC(2) (p=0.007). Our results support the idea that Rho-123 is the substrate of choice for leukemic cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Corantes Fluorescentes/metabolismo , Leucemia/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Carbocianinas/metabolismo , Citometria de Fluxo , Humanos , Leucemia/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Rodamina 123/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Since the first morphological description of the gap junctions use electron microscopy, a considerable number of techniques has been introduced to evaluate gap junction channel functionality, many of which use dye transfer techniques, such as dye injection and fluorescent dye transfer, analyzed by flow cytometry. METHODS: To analyze dye transfer, generally one population of cells is incubated with calcein-AM (0.5 microM) for 30 min at 37 degrees C, and the other population was incubated with the lipophilic dye DiIC(18) (3) (10 microM) for 1 h at 37 degrees C; after incubation, these cells were washed five times with PBS and cocultured for different times, and then the dye transfer was analyzed by flow cytometry. RESULTS: In this short overview, we focus on some advantages and disadvantages of flow cytometry as a technique to investigate gap junction-mediated intercellular communication (GJIC). In addition, we point out some technical pitfalls that we have encountered when applying this technique to study gap junctions in immune system cells. CONCLUSIONS: Analysis of fluorescent dye transfer by flow cytometry is a useful tool to investigate GJIC. However, some points must be taken into consideration before using this methodology, which are discussed herein.