RESUMO
S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-gamma, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.
Assuntos
Anti-Inflamatórios não Esteroides/imunologia , Calgranulina A/imunologia , Calgranulina B/imunologia , Sequestradores de Radicais Livres/imunologia , Leucócitos/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/imunologia , Calgranulina A/química , Calgranulina B/química , Cisteína/química , Cisteína/imunologia , Células Endoteliais/imunologia , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Hemoglobinas/imunologia , Humanos , Inflamação/imunologia , Interferon gama/farmacologia , Interleucina-10/imunologia , Lipopolissacarídeos/farmacologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação/imunologia , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Oxidantes/imunologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Both nitrogen monoxide (NO) and carbon monoxide (CO) are biologically relevant diatomic effector molecules that mediate a variety of biological functions through their avid binding to iron (Fe). Previous studies showed that NO can inhibit Fe uptake from transferrin (Tf) and increase Fe mobilisation from cells [J. Biol. Chem. 276 (2001) 4724]. We used CO gas, a CO-generating agent ([Ru(CO)3Cl2]2), and cells stably transfected with the CO-producing enzyme, haem oxygenase 1 (HO1), to assess the effect of CO on Fe metabolism. These results were compared to the effects of NO produced by a variety of NO-generating agents, including S-nitrosoglutathione (GSNO), spermine-NONOate (SperNO) and S-nitroso-N-acetylpenicillamine (SNAP). Incubation of cells with CO inhibited 59Fe uptake from 59Fe-Tf by cells, and like NO, reduced ATP levels. Hence, the ability of both agents to inhibit 59Fe uptake may be partially mediated by inhibition of energy-dependent processes. These results showing a CO-mediated decrease in 59Fe uptake from 59Fe-Tf using exogenous CO were in agreement with studies implementing cells transfected with HO1. Like NO, CO markedly prevented 59Fe uptake into ferritin. In comparison to the avid ability of exogenous CO to inhibit 59Fe uptake, it had less effect on cellular 59Fe mobilisation. Experiments with HO1-transfected cells compared to control cells showed that 59Fe mobilisation was slightly enhanced. In contrast to NO, CO did not affect the RNA-binding activity of the iron regulatory protein 1 that plays an important role in Fe homeostasis. Our studies demonstrate that subtle differences in the chemistry of NO and CO results in divergence of their ability to affect Fe metabolism.
Assuntos
Monóxido de Carbono/metabolismo , Ferro/metabolismo , Óxido Nítrico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Ferritinas/metabolismo , Fibroblastos/metabolismo , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Humanos , Proteínas de Membrana , Camundongos , Nitritos/metabolismo , RNA/metabolismo , Fatores de Tempo , Transfecção , Transferrina/metabolismoRESUMO
PURPOSE: The development of novel and potent iron chelators as clinically useful antitumor agents is an area of active interest. Antiproliferative activity of chelators often relates to iron deprivation or stimulation of iron-dependent free radical damage. Recently, we showed that novel iron chelators of the di-2-pyridylketone isonicotinoyl hydrazone (PKIH) class have potent and selective antineoplastic activity (E. Becker, et al., Br. J. Pharmacol., 138: 819-30, 2003). In this study, we assessed the effects of the PKIH analogues on the redox activity of iron in terms of understanding their antitumor activity. EXPERIMENTAL DESIGN: We tested the PKIH analogues for their ability to promote iron-mediated ascorbate oxidation, benzoate hydroxylation, and plasmid degradation. Subsequent experiments assessed their ability to bind DNA, inhibit topoisomerase I, and cause DNA damage. To measure intracellular reactive oxygen species, we used the redox-sensitive probe, 2',7'-dichloro-fluorescein-diacetate, to measure intracellular PKIH-dependent redox activity. RESULTS: The PKIH analogues had relatively little effect on ascorbate oxidation in the presence of Fe(III) but stimulated benzoate hydroxylation and plasmid DNA degradation in the presence of Fe(II) and H2O2. These ligands could not inhibit DNA topoisomerase I or cause DNA damage in intact cells. PKIH markedly increased the intracellular generation of reactive oxygen species, and this was inhibited by catalase. This enzyme also decreased the antiproliferative effect of PKIH, indicating H2O2 played a role in its cytotoxic activity. CONCLUSIONS: Our results suggest that the antiproliferative effects of these chelators relates to intracellular iron chelation, followed by the stimulation of iron-mediated free radical generation via the so-formed iron complex.
Assuntos
Antineoplásicos/farmacologia , Quelantes/farmacologia , Radicais Livres , Hidrazonas/farmacologia , Peróxido de Hidrogênio/farmacologia , Ferro/metabolismo , Ferro/farmacologia , Piridinas/farmacologia , Ácido Ascórbico/metabolismo , Azóis/farmacologia , Catalase/metabolismo , Linhagem Celular Tumoral , Corantes/farmacologia , DNA/metabolismo , Dano ao DNA , DNA Topoisomerases Tipo I/química , Relação Dose-Resposta a Droga , Humanos , Peróxido de Hidrogênio/química , Isoindóis , Ligantes , Modelos Químicos , Compostos Organosselênicos/farmacologia , Oxirredução , Oxigênio/metabolismo , Plasmídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologiaRESUMO
PURPOSE: Tumors are sensitive to iron (Fe) chelation therapy with the clinically used chelator desferrioxamine (DFO). Recently, the potent inhibitor of ribonucleotide reductase, Triapine, has entered clinical trials as an anticancer agent. This compound is a potential Fe chelator, but despite this, no investigations have examined its effect on cellular Fe metabolism. This is essential for understanding its mechanism of action and clinical effects. EXPERIMENTAL DESIGN: We compared the effect of Triapine with DFO, and also with the novel Fe chelator, 311, which shows marked antiproliferative activity. This latter ligand was relevant to compare, because it is tridentate like Triapine and shares structural similarity. We assessed the effects of chelators on proliferation, Fe uptake, Fe efflux, the expression of cell cycle control molecules, and iron-regulatory protein-RNA-binding activity. Redox activity was determined by ascorbate oxidation, benzoate hydroxylation, plasmid DNA degradation, and the precipitation of cellular DNA. These studies have been performed using several neuroepithelioma and neuroblastoma cell lines and a variety of normal cell types including fibroblasts, umbilical vein endothelial cells, skeletal muscle cells, monocyte-derived macrophages, and bone marrow stem cells. RESULTS: Triapine was twice as effective as DFO at mobilizing (59)Fe from prelabeled cells but was much less efficient than 311. In terms of preventing (59)Fe uptake from Tf, Triapine and DFO had similar activity, having far less efficacy than 311. All three of the chelators showed greater activity against the proliferation of neoplastic than of normal cells, the effect of 311 and Triapine being similar and these two chelators being significantly (P < 0.0001) more active than DFO. Complexation of Triapine with Fe had no appreciable effect on its antiproliferative activity, whereas addition of Fe totally inhibited the effects of DFO and 311. Furthermore, the Triapine Fe complex was shown to be redox active. CONCLUSION: The cytotoxic mechanism of action of Triapine was different from that of DFO and 311, with the combined action of Fe chelation and free radical generation being involved.
Assuntos
Quelantes/farmacologia , Desferroxamina/farmacologia , Ferro/metabolismo , Isoniazida/farmacologia , Piridinas/farmacologia , Tiossemicarbazonas/farmacologia , Animais , Ácido Ascórbico/metabolismo , Northern Blotting , Western Blotting , Divisão Celular/efeitos dos fármacos , Quelantes/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Glutationa/metabolismo , Humanos , Ferro/farmacocinética , Quelantes de Ferro/farmacologia , Modelos Químicos , Oxirredução , Oxigênio/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Ratos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron (Fe) release. We have shown that NO-mediated Fe efflux from cells required glutathione (GSH), and we have hypothesized that a GS-Fe-NO complex was released. Hence, we studied the role of the GSH-conjugate transporter multidrug resistance-associated protein 1 (MRP1) in NO-mediated Fe efflux. MCF7-VP cells overexpressing MRP1 exhibited a 3- to 4-fold increase in NO-mediated 59Fe and GSH efflux compared with WT cells (MCF7-WT) over 4 h. Similar results were found for other MRP1-overexpressing cell types but not those expressing another drug efflux pump, P-glycoprotein. NO-mediated 59Fe and GSH efflux were temperature- and energy-dependent and were significantly decreased by the GSH-depleting agent and MRP1 transport inhibitor L-buthionine-[S,R]-sulfoximine. Other MRP1 inhibitors, MK571, probenecid, and difloxacin, significantly inhibited NO-mediated 59Fe release. EPR spectroscopy demonstrated the dinitrosyl-dithiol-Fe complex (DNIC) peak in NO-treated cells was increased by MRP1 inhibitors, indicating inhibited DNIC transport from cells. The extent of DNIC accumulation correlated with the ability of MRP1 inhibitors to prevent NO-mediated 59Fe efflux. MCF7-VP cells were more sensitive than MCF7-WT cells to growth inhibition by effects of NO, which was potentiated by L-buthionine-[S,R]-sulfoximine. These data indicate the importance of GSH in NO-mediated inhibition of proliferation. Collectively, NO stimulates Fe and GSH efflux from cells via MRP1. Active transport of NO by MRP1 overcomes diffusion that is inefficient and nontargeted, which has broad ramifications for understanding NO biology.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Glutationa/metabolismo , Ferro/metabolismo , Óxido Nítrico/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Butionina Sulfoximina/metabolismo , Linhagem Celular , Proliferação de Células , Inibidores Enzimáticos/metabolismo , Humanos , Radioisótopos de Ferro/metabolismo , Camundongos , Doadores de Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/metabolismo , Espermina/análogos & derivados , Espermina/metabolismo , Temperatura , Transferrina/metabolismoRESUMO
Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/efeitos dos fármacos , Isoflavonas/farmacologia , Melanoma/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Caspases/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Isoflavonas/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Conformação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/efeitos dos fármacosRESUMO
Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.
Assuntos
Ferritinas/metabolismo , Glutationa/metabolismo , Ferro/metabolismo , Óxido Nítrico/farmacologia , Penicilamina/análogos & derivados , Espermina/análogos & derivados , Adenocarcinoma/patologia , Animais , Neoplasias da Mama/patologia , Permeabilidade da Membrana Celular , Sistema Livre de Células , Citosol/metabolismo , Desferroxamina/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Quelantes de Ferro/farmacologia , Ativação de Macrófagos , Camundongos , Neuroblastoma/patologia , Tumores Neuroectodérmicos Primitivos Periféricos/patologia , Doadores de Óxido Nítrico/farmacologia , Óxidos de Nitrogênio , Oxirredução , Penicilamina/farmacologia , S-Nitrosoglutationa/farmacologia , Espermina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Many effector functions of nitrogen monoxide (NO) and carbon monoxide (CO) are mediated through their high-affinity for iron (Fe). In this review, the roles of NO and CO are examined in terms of their effects on the molecular and cellular mechanisms involved in Fe metabolism. Both NO and CO avidly form complexes with a plethora of Fe-containing molecules. The generation of NO and CO is mediated by the nitric oxide synthase and haem oxygenase (HO) families of enzymes respectively. The effects of NO on Fe metabolism have been well characterized, whereas knowledge of the effects of CO remains within its infancy. In terms of the role of NO in Fe metabolism, one of the best characterized interactions includes its effect on the iron regulatory proteins. These molecules are mRNA-binding proteins that control the expression of the transferrin receptor 1 and ferritin, molecules that are involved in Fe uptake and storage respectively. Apart from this, activated macrophages impart their cytotoxic activity by generating NO, which results in marked Fe mobilization from tumour-cell targets. This deprives the cell of the Fe that is required for DNA synthesis and energy production. Considering that HO degrades haem, resulting in the release of CO, Fe(II) and biliverdin, it is suggested that a CO-Fe complex will form. This may account for the rapid Fe mobilization observed from macrophages after haemoglobin catabolism. Intriguingly, overexpression of HO results in cellular Fe mobilization, suggesting that CO has a similar effect to NO on Fe trafficking. Preliminary evidence suggests that, like NO, CO plays important roles in Fe metabolism.