RESUMEN
Secreted quiescin/sulfhydryl oxidase (QSOX) is overexpressed in many tumor cell lines, including melanoma, and is usually associated with a pro-invasive phenotype. Our previous work described that B16-F10 cells enter in a quiescent state as a protective mechanism against damage generated by reactive oxygen species (ROS) during melanogenesis stimulation. Our present results show that QSOX activity was two-fold higher in cells with stimulated melanogenesis when compared to control cells. Considering that glutathione (GSH) is one of the main factor responsible for controlling redox homeostasis in cells, this work also aimed to investigate the relationship between QSOX activity, GSH levels and melanogenesis stimulation in B16-F10 murine melanoma cell line. The redox homeostasis was impaired by treating cells with GSH in excess or depleting its intracellular levels through BSO treatment. Interestingly, GSH-depleted cells without stimulation of melanogenesis kept high levels of viability, suggesting a possible adaptive mechanism of survival even under low GSH levels. They also showed lower extracellular activity of QSOX, and higher QSOX intracellular immunostaining, suggesting that this enzyme was less excreted from cells and corroborating with a diminished extracellular QSOX activity. On the other hand, cells under melanogenesis stimulation showed a lower GSH/GSSG ratio (8:1) in comparison with control (non-stimulated) cells (20:1), indicating a pro-oxidative state after stimulation. This was accompanied by decreased cell viability after GSH-depletion, no alterations in QSOX extracellular activity, but higher QSOX nucleic immunostaining. We suggest that melanogenesis stimulation and redox impairment caused by GSH-depletion enhanced the oxidative stress in these cells, contributing to additional alterations of its metabolic adaptive response.
Asunto(s)
Melanoma , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Animales , Ratones , Glutatión/metabolismo , Melanoma/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismoRESUMEN
The use of nanoparticles as drug delivery systems to simultaneously carry several therapeutic agents is an attractive idea to create new synergic treatments and to develop the next generation of cancer therapies. Therefore, the goal of this study was the simultaneous encapsulation of a hydrophilic drug, sodium diethyldithiocarbamate (DETC), and a hydrophobic drug, 4-nitrochalcone (4NC), in beeswax nanoparticles (BNs) to evaluate the in vitro synergic activity of this combination against melanoma (B16F10) cells. BNs were prepared by water/oil/water double emulsion in the absence of organic solvents. Transmission electron microscopy imaging and dynamic light scattering analyses indicated the formation of BNs with a semispherical shape, average diameter below 250 nm, relatively narrow distributions, and negative zeta potential. The double emulsion technique proved to be effective for the simultaneous encapsulation of DETC and 4NC with efficiencies of 86.2% and 98.7%, respectively, and this encapsulation did not affect the physicochemical properties of the BNs. DETC and 4NC loaded in BNs exhibited a higher cytotoxicity toward B16F10 cells than free 4NC and DETC. This simultaneous encapsulation led to a synergic effect of DETC and 4NC on B16F10 cells, decreasing the cell viability from 46% (DETC BNs) and 54% (4NC BNs) to 64% (DETC+4NC BNs). Therefore, the IC50 of DETC+4NC was also lower than that of either when individually encapsulated, and that of free DETC or 4NC. Therefore, DETC and 4NC were efficiently simultaneously encapsulated in BNs and this drug combination was able to generate an in vitro synergic therapeutic effect on B16F10 cells.
Asunto(s)
Melanoma , Nanopartículas , Ditiocarba , Portadores de Fármacos , Humanos , Tamaño de la Partícula , CerasRESUMEN
The combination of hyperthermia and chemotherapy has a potential synergic effect in antitumor activity. The development of new biocompatible and biodegradable polymers to simultaneously encapsulate magnetic nanoparticles (MNPs) and antitumoral drugs offer new cancer treatment opportunities. Here, biodegradable and biocompatible poly(thioether-ester) (PTEe) was used to encapsulate MNPs and 4-nitrochalcone (4NC) using miniemulsification and solvent evaporation. The resulting hybrid particles (MNPs-4NC-PTEe) had nanometer-scale diameters, spherical morphology, negative surface charge, high encapsulation efficiency, and superparamagnetic properties. Results showed that 4NC release occurred through diffusion. Free 4NC and MNPs + 4NC-PTEe did not have any cytotoxic effect on erythrocytes and mouse embryonic fibroblast (NIH3T3) cells. 4NC antitumor activity was verified on human cervical cancer (HeLa) and melanoma (B16F10) cells. Cellular uptake of MNPs + 4NC-PTEe nanoparticles was higher in HeLa cells compared to B16F10 and NIH3T3 cells. The hyperthermia application (115 kHz-500 Oe) potentiated the 4NC effects on HeLa and B16F10 cells when MNPs + 4NC-PTEe nanoparticles were used, indicating more effective antitumor activity. We concluded that the use of MNPs + 4NC-PTEe nanoparticles associated with hyperthermia is a promising form of treatment for some types of cancers.
Asunto(s)
Hipertermia Inducida , Nanopartículas de Magnetita , Nanopartículas , Animales , Ésteres , Fibroblastos , Células HeLa , Humanos , Hipertermia , Ratones , Células 3T3 NIH , SulfurosRESUMEN
Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.
Asunto(s)
Ácido Fólico/química , Ácido Gálico/análogos & derivados , Nanopartículas de Magnetita/química , Polimetil Metacrilato/química , Animales , Materiales Biocompatibles , Línea Celular , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/farmacología , Endocitosis , Eritrocitos/citología , Ácido Gálico/farmacocinética , Células HeLa , Hemólisis , Humanos , Cinética , Ratones , Tamaño de la Partícula , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Temperatura , TermogravimetríaRESUMEN
The present study investigated in vitro the effects of sulphated heterorhamnan (Go3), iota-/nu-carrageenans (G3d and EHW-I) and arabinogalactan (ARAGAL) polysaccharides on macrophage activation and inhibition of intracellular amastigotes of Leishmania (L.) amazonensis. All the sulphated polysaccharides (Go3, G3d and EHW-I) promoted increased nitric oxide production varying from 71 to 110%. The leishmanicidal activity of all compounds was compared to the inhibition effect of Meglumine Antimoniate at 300µg/mL (â¼79%), used as positive control. Inhibition of Leishmania (L.) amazonensis growth was 55% with 5µg/mL of Go3, 50% and 98% to G3d and EHW-I, respectively at 10µg/mL, and 88% with 10µg/mL of ARAGAL. The superoxide anion scavenging activity for the sulphated polysaccharides varied from approximately 30-55% at 10µg/mL. In conclusion, the results of the present study indicate the promising potential of these polysaccharides for the development of new alternative therapeutic agents against leishmaniasis.
Asunto(s)
Leishmania/efectos de los fármacos , Polisacáridos/química , Polisacáridos/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Animales , Citotoxicidad Inmunológica/efectos de los fármacos , Leishmania/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Ratones , Óxido Nítrico/biosíntesis , Pruebas de Sensibilidad Parasitaria , Superóxidos/metabolismoRESUMEN
In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 µM after 24-h treatment, whereas MI-D required a 50 µM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 µM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 µM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 µM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Tiadiazoles/química , Tiadiazoles/farmacología , Animales , Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Células Hep G2 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Masculino , Ratas , Ratas Wistar , Tiadiazoles/efectos adversosRESUMEN
Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50 µM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50 µM) did not affect the viability of hepatocytes in suspension after 1-40 min of incubation. However, the viability of the cultured hepatocytes was decreased by â¼66% as a consequence of treatment with SYD-1 (50 µM) for 18 h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by â¼19%. The morphological changes in the cultured cells treated with SYD-1 (50 µM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50 µM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by â¼36%, whereas the lactate levels were increased by â¼22% (for the 50 µM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by â¼79% and â¼51%, respectively, by SYD-1 (50 µM). In these cells, SYD-1 (50 µM) increased the pyruvate and lactate levels by â¼84% and â¼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension.
Asunto(s)
Hepatocitos/efectos de los fármacos , Oxadiazoles/farmacología , Animales , Antineoplásicos/farmacología , Células Cultivadas , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Ácido Láctico/metabolismo , Masculino , Oxadiazoles/química , Ácido Pirúvico/metabolismo , Ratas , Ratas WistarRESUMEN
Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated ß-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Simvastatina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Senescencia Celular/fisiología , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Humanos , Melanoma/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75µmolmg(-1) protein) inhibited the lipoperoxidation induced by Fe(3+)/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0µmolmg(-1) protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α'-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25-1.0µmolmg(-1) protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9-19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25-1.0µmolmg(-1) protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca(2+)/phosphate by approximately 30% (1.0µmolmg(-1) protein). When Ca(2+)/H2O2 were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0µmolmg(-1) of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.
Asunto(s)
Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Oxadiazoles/farmacología , Estrés Oxidativo/fisiología , Animales , Catalasa/metabolismo , Fluorometría , Masculino , Mitocondrias Hepáticas/enzimología , Oxadiazoles/metabolismo , Oxidación-Reducción , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
In this study, the responses of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX) in mitochondria from embryogenic cells of A. angustifolia subjected to cold stress (4°C for 24 h or 48 h) is reported. In the mitochondria of stressed cells, PUMP activity increased by approximately 45% (at 24h and 48 h), which was determined by measuring the oxygen consumption after the addition of linoleic acid and the inhibition by BSA and ATP. PUMP activation was confirmed using transmembrane electrical potential (Δψ) assays. Immunoblot assays showed an increase of PUMP expression by 40% and 150% after 24h and 48 h of cold stress, respectively. AOX activity, measured under conditions similar to those of the PUMP assays, was only slightly increased in the mitochondria from stressed cells (at 24h and 48 h), as demonstrated by oxygen consumption experiments. Cell viability was unaffected by cold stress, indicating that the effects on PUMP and AOX were not caused by cell death. These results show that the main response of this gymnosperm to cold stress is the activation of PUMP, which suggests that this protein may be involved in the control of reactive oxygen species generation, which has been previously associated with this condition.
Asunto(s)
Canales Iónicos/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Proteínas de Plantas/metabolismo , Tracheophyta/fisiología , Técnicas de Cultivo de Célula , Supervivencia Celular , Frío , Mitocondrias/metabolismo , Estrés Fisiológico , Tracheophyta/anatomía & histología , Tracheophyta/embriología , Tracheophyta/enzimología , Proteína Desacopladora 1RESUMEN
Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen ((1)O(2)), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with (1)O(2), they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT.
Asunto(s)
Daño del ADN , Reparación del ADN , Oxígeno Singlete/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Ferritinas/metabolismo , Melaninas/fisiologíaRESUMEN
The aim of this work was to assess the significance of the interaction of the 1,3,4-thiadiazolium derivatives MI-J, MI-4F and MI-2,4diF with mitochondrial membrane and their effects on energy-linked functions. Mitochondrial swelling in the absence of substrate was inhibited by all derivatives; however, the fluorine derivatives were most effective. MI-4F decreased swelling by ~32% even at the lowest concentration (65 nmol mg(-1) protein), reaching ~67% at the concentration of 130 nmol mg(-1) protein. Swelling of mitochondria in the presence of oxidizable substrates was also strongly decreased by all derivatives. This effect was more pronounced when using glutamate plus malate, and also fluorine derivatives, which promoted complete inhibition at all concentrations (6.5-130 nmol mg(-1) protein). Swelling occurred when succinate was the substrate in the presence of MI-J (6.5-65 nmol mg(-1) protein); however, the shrinkage rate was strongly decreased. MI-4F and MI-2,4diF also inhibited swelling, with total inhibition occurring at a concentration of 65 nmol mg(-1) protein. Lipid peroxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria was inhibited time- and dose-dependently by the derivatives, reaching complete inhibition at the highest concentration (80 nmol mg(-1) protein). However, when lipid peroxidation was initiated by peroxyl radicals generated from AAPH, the inhibition was less intense, reaching ~50%, ~40% and ~58% with MI-J, MI-4F and MI-2,4diF (80 nmol mg(-1) protein), respectively. The mesoionic compounds also showed superoxide radical scavenging ability of ~22%, ~32% and ~40% (80 nmol mg(-1) protein), respectively. Fluorescence polarization experiments showed that the derivatives are able to enter the bilayer, decreasing its fluidity in the hydrophobic DMPC membrane region and ordering the fluid phase. Our results suggest that MI-J, MI-4F and MI-2,4diF interact significantly, albeit in different modes, with mitochondrial membrane, and that fluorine derivatives seem to alter the membrane's properties more markedly.
Asunto(s)
Depuradores de Radicales Libres/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Tiadiazoles/farmacología , Animales , Polarización de Fluorescencia , Masculino , Fluidez de la Membrana/efectos de los fármacos , Fluidez de la Membrana/fisiología , Mitocondrias Hepáticas/fisiología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/fisiología , Dilatación Mitocondrial/efectos de los fármacos , Dilatación Mitocondrial/fisiología , Ratas , Ratas Wistar , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Flavonoids are a large group of polyphenolic compounds that have received considerable attention because of their biological and physiological importance. The flavone (2-phenyl-4H-1-benzopyran-4one) used in this work is found in some cereal grains and generates several biological activities, including: apoptosis induction, cell cycle arrest, caspase activation and inhibition of tumor cell proliferation. However, its effects on the hepatic mitochondrial metabolism are still unknown. We evaluated the effect of flavone on the metabolism of mitochondria isolated from rat liver. Polarographic experiments using 200 micromol L(-1) flavone and rat liver mitochondria oxidizing glutamate or succinate indicated that both substrates underwent: (i) reduction of state 3 respiration; (ii) stimulation of state 4 respiration; (iii) reduction of the respiratory control coefficient; and (iv) reduction of the ADP/O ratio. An analysis of the activity of enzymatic complexes in the respiratory chain showed that flavone acts between complexes I and III. Flavone reduced the membrane electric potential at doses of 100, 150 and 200 micromol L(-1). Flavone at certain doses (75-200 micromol L(-1)) reduced mitochondrial swelling in the presence of valinomycin and KNO(3), suggesting that flavone could induce changes in mitochondrial membrane properties. These results demonstrate that the inhibition of mitochondrial enzymes in the respiratory chain coupled with the effects on membrane properties are promoted by the core structure of flavones, and these effects may be in part responsible for the cytotoxic effects of flavones.
Asunto(s)
Transporte de Electrón/efectos de los fármacos , Flavonas/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Flavonas/química , Masculino , Mitocondrias Hepáticas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The main goal of this work was to investigate the relationship between the effects of three new 1,3,4-thiadiazolium mesoionic derivatives on mitochondrial bioenergetics and their previously described chemical structure and antimelanoma activity. The 4-phenyl-5-(2'-Y, 4'-X or 4'-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides differed from each other only in the cinnamoyl ring substituent: MI-J, X=OH; MI-F, X=F; MI-2,4diF X=Y=F. The state 3 respiratory rate was strongly decreased by all derivatives, reaching total inhibition of MI-4F and MI-2,4diF (130nmolmg(-1) protein), when glutamate plus malate were used as substrate. State 3 inhibition was less accentuated with succinate as substrate. Analyses of segments of the respiratory chain indicated complexes I and IV as sites inhibited by the derivatives. State 4 respiration was strongly stimulated by the three derivatives, and was characterized as an uncoupling effect, which was more intense for MI-4F. This stimulus was so pronounced that the values of RCC and ADP/O ratio were only calculated for the lowest concentration (6.5nmolmg(-1) protein). In intact mitochondria, the ATPase activity was increased dramatically by approximately 120%, approximately 207% and approximately 261% for MI-J, MI-4F and MI-2,4diF (32.5nmolmg(-1) protein), respectively. In conclusion, the presence of fluorine substituent in the cinnamoyl ring intensifies the effect of mesoionic compounds on mitochondrial functions and, in this context, hydrophobicity is more important than the electronic effect, which was correlated to antimelanoma activity described previously for these compounds.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Tiadiazoles/química , Tiadiazoles/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Masculino , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Wistar , Frecuencia Respiratoria/efectos de los fármacosRESUMEN
Important biological activities have been described for mesoionic compounds. We previously reported that MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride) inhibited the respiratory chain, collapsed the transmembrane potential, and stimulated ATPase activity in intact rat liver mitochondria. It is known that drugs that affect mitochondrial membrane potential may facilitate the induction of cell death by apoptosis. Mitochondria have also a central role in the generation of reactive oxygen species, therefore it would be important to investigate how MI-D could affect processes related to oxidative stress. In this work, we evaluated the effects of MI-D on the lipoperoxidation and its ability to scavenge free radicals. Interestingly, it was observed that MI-D promoted a strong inhibition of the lipoperoxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria (95%+/-0.27 at the highest concentration of 80 nmol mg(-1) protein) in a dose-dependent manner. However, at the same concentration its effect was less intense (22%+/-3.46) when the lipoperoxidation was initiated by peroxyl radicals generated from the azocompound AAPH. Lipid peroxidation in both coupled and uncoupled submitochondrial particles initiated with Fe(2+)/NADH was also inhibited by MI-D. The inhibition was about four times greater in coupled particles (approximately 34% at 80 nmol mg(-1) protein) in relation to uncoupled. MI-D inhibited the soybean phosphatidylcholine liposomes lipoperoxidation in a dose-dependent manner (5-80 microM) regardless of the radical being generated in lipid or aqueous phase. The mesoionic compound showed ability of scavenging superoxide radical (7, 11 and 31% for 25, 38 and 80 microM, respectively). Our results strongly suggest that the inhibition of lipoperoxidation promoted by MI-D is due to its scavenger action and to its previously described uncoupling effect.
Asunto(s)
Cinamatos/farmacología , Depuradores de Radicales Libres/farmacología , Peroxidación de Lípido/efectos de los fármacos , Tiadiazoles/farmacología , Amidinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Liposomas/antagonistas & inhibidores , Liposomas/metabolismo , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfatidilcolinas/antagonistas & inhibidores , Fosfatidilcolinas/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 +/- 1.56 and 4.05 +/- 1.99 nmol NO per 10(7) cells, respectively). When isolated from a 6-day-old tumor, a significantly lower production of IL-6 and higher production of TNF-alpha than in cells from a 4-day-old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE(2) by Walker 256 cells isolated from the 6-day-old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time.
Asunto(s)
Líquido Ascítico/patología , Caquexia/metabolismo , Carcinoma 256 de Walker/complicaciones , Animales , Arginasa/metabolismo , Poliaminas Biogénicas/metabolismo , Caquexia/etiología , Carcinoma 256 de Walker/patología , Línea Celular Tumoral , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Masculino , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismo , Urea/metabolismoRESUMEN
An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.
Asunto(s)
Mitocondrias Hepáticas/efectos de los fármacos , Oxadiazoles/farmacología , Sidnonas/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Respiración de la Célula/efectos de los fármacos , Iones/química , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Dilatación Mitocondrial/efectos de los fármacos , Estructura Molecular , Oligomicinas/farmacología , Oxadiazoles/síntesis química , Ratas , Ratas Wistar , Sidnonas/químicaRESUMEN
Bisphenols are a class of compounds that exhibit a broad spectrum of antimicrobial activity. One of the most widely used member of this group is triclosan (TRN). TRN is a synthetic, non-ionic, broad-spectrum antimicrobial agent, which is incorporated into several products, including hand soaps and detergents and those of skin care and oral hygiene. The effects of TRN on mitochondrial respiratory parameters and the inner mitochondrial membrane potential (DeltaPsi) are described. That of TRN (up to 60 nmol mg(-1) protein) on isolated liver mitochondria decreased oxygen consumption of state 3 respiration, as well as DeltaPsi, but increased oxygen consumption of state 4 respiration, characteristic of an uncoupler effect. Analysis of segments of the respiratory chain suggested that the TRN inhibition site is located between complexes II and III. Mitochondrial swelling, energized or driven by the K+ diffusion potential using valinomycin, was also inhibited by TRN, the former being completely inhibited at concentrations greater than 10 nmol TRN mg(-1) protein, suggesting that it is also able to interfere with fluidity of the inner mitochondrial membrane. These results suggest that, besides its antibacterial effect, TRN can also impair the mitochondrial function of animal cells.
Asunto(s)
Antiinfecciosos Locales/farmacología , Hígado/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Triclosán/farmacología , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Relación Dosis-Respuesta a Droga , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/fisiología , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Ratas , Ratas Wistar , Desacopladores/farmacologíaRESUMEN
The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.
Asunto(s)
Citrinina/farmacología , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Quelantes del Hierro/farmacología , Ácido Ascórbico/metabolismo , Citrinina/metabolismo , Ditiotreitol/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ácido Edético/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Ferrozina/metabolismo , Radicales Libres/metabolismo , Peróxido de Hidrógeno/metabolismo , Quelantes del Hierro/metabolismo , Cinética , Ligandos , Peroxidación de Lípido/efectos de los fármacos , Oxidación-Reducción , Oxígeno/metabolismo , Fenantrolinas/metabolismo , Espectrofotometría UltravioletaRESUMEN
Em alguns tipos de porfirias como porfiria aguda intermitente (PAI) e tirosinemia hereditária tipo (HT1) observa-se acúmulo do ácido 5-aminolevulínico (ALA) no sangue e tecidos. In vitro, o ALA sobre oxidaçäo pelo oxigênio molecular, catalisada por complexos de ferro, gerando o ácido 4,5-dioxovalérico (DOVA), íons NH4+, H2O2 e os radicais ALA, O2, e HO. ALA é capaz também de causar lesöes oxidativas a várias biomoléculas e inclusive induzir a liberaçäo de ferro de ferritina. Sendo assim, o ALA poderia agir como um pró-oxidante endógeno. Nossos estudos foram realizados com o objetivo de: 1) verificar a possibilidade de ocorrer processo autocatalítico durante a oxidaçäo do ALA em presença de ferritina de baço de cavalo (HoSF) e observar a influência de fosfato neste processo...