Iron chelators inhibit the growth and induce the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors.
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Iron is suspected to be involved in the induction and/or progression of various human tumors. More particularly, iron may be involved in the pathogenesis of Kaposi's sarcoma, a tumor of probable vascular origin. This study was designed to investigate the effect of iron deprivation on Kaposi's sarcoma. The effects of iron chelators and iron deprivation associated with serum withdrawal were investigated on Kaposi's sarcoma-derived spindle cells, on a transformed Kaposi's sarcoma cell line (Kaposi's sarcoma Y-1) and on endothelial cells, which are the probable progenitors of Kaposi's sarcoma cells. Desferrioxamine and deferiprone, two chemically unrelated iron chelators, induced a time- and concentration-dependent inhibition of endothelial and Kaposi's sarcoma cell growth. The inhibition of cell growth was associated with a decrease in Ki-67 and in both stable and total proliferating cell nuclear antigen expression. Inhibition of the progression through the G1-phase of the cell cycle was further evidenced by decreased expression of cyclin D1 and of p34 cyclin-dependent kinase 4. Terminal deoxynucleotidyl transferase-mediated desoxyuridinetriphosphate nick end labeling assay, flow cytometry with annexin-V-fluorescein and morphologic analysis indicated that iron chelation also induced a time- and concentration-dependent apoptosis. This apoptotic effect was prevented by the addition of exogenous iron. Induction of iron deprivation in the culture medium by serum withdrawal led to similar cell cycle effects, which, however, could only be partly reverted by the addition of exogenous iron. In conclusion, these results show that iron deprivation inhibits the growth and induces the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors. This suggests that iron chelators may represent a potential therapeutic approach for the treatment of Kaposi's sarcoma. (+info)
Extracellular iron acquisition by mycobacteria: role of the exochelins and evidence against the participation of mycobactin.
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Mycobacterium bovis var. BCG was grown under iron-deficient conditions in the presence and absence of 1% Tween 80. Mycobactin, the iron iron ionophore of mycobacteria, was found solely within the bacteria grown in the absence of Tween, but low concentrations (0.75 mug/ml) of it appeared in the medium in the presence of the surfactant. Both types of medium contain agents, named exochelins, which could solubilize iron. 55Fe added to spent culture media was recovered only chelated to these compounds. Two exochelins were detected, isolated, and purified. Neither were precursors or breakdown products of mycobactin. In the desferri-form, exochelin MB-2, the major component, reversed the inhibitory effect of serum on the growth of BCG, and in their ferri-forms exochelins MB-1, MB-2, and MS (from Mycobacterium smegmatis) stimulated the growth of their producing organism in the presence of serum. Exochelin MB-2 could physically remove iron from ferritin, and BCG used ferritin as a source of iron during growth even when ferritin was separated from the bacteria by a dialysis membrane. As solutions of the exochelins were freely dialyzable, whereas solutions of mycobactin, even in Tween, were not, only exochelin could have been active in this experiment. The exochelins are proposed as the functional extracellular iron-binding agents of BCG and other mycobacteria, the role of mycobactin being confined to that of a cell wall iron transporter. (+info)
The overlapping angB and angG genes are encoded within the trans-acting factor region of the virulence plasmid in Vibrio anguillarum: essential role in siderophore biosynthesis.
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Products encoded in the trans-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of Vibrio anguillarum. Here we identify angB, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis. We demonstrate that a 287-amino-acid polypeptide, encoded by angB and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2, 3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate. Complementation of various angB mutations provided evidence that an additional, overlapping gene exists at this locus. This second gene, designated angG, also has an essential biosynthetic function. The angG gene directs the expression of three polypeptides when overexpressed in Escherichia coli, all of which are translated in the same frame as AngB. The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule. Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the angB and angG genes. (+info)
Nitrogen monoxide (no) and glucose: unexpected links between energy metabolism and no-mediated iron mobilization from cells.
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Nitrogen monoxide (NO) affects cellular iron metabolism due to its high affinity for this metal ion. Indeed, NO has been shown to increase the mRNA binding activity of the iron-regulatory protein 1, which is a major regulator of iron homeostasis. Recently, we have shown that NO generators increase (59)Fe efflux from cells prelabeled with (59)Fe-transferrin (Wardrop, S. L., Watts, R. N., and Richardson, D. R. (2000) Biochemistry 39, 2748-2758). The mechanism involved in this process remains unknown, and in this investigation we demonstrate that it is potentiated upon adding d-glucose (d-Glc) to the reincubation medium. In d-Glc-free or d-Glc-containing media, 5.6 and 16.5% of cellular (59)Fe was released, respectively, in the presence of S-nitrosoglutathione. This difference in (59)Fe release was observed with a variety of NO generators and cell types and was not due to a change in cell viability. Kinetic studies showed that d-Glc had no effect on the rate of NO production by NO generators. Moreover, only the metabolizable monosaccharides d-Glc and d-mannose could stimulate NO-mediated (59)Fe mobilization, whereas other sugars not easily metabolized by fibroblasts had no effect. Hence, metabolism of the monosaccharides was essential to increase NO-mediated (59)Fe release. Incubation of cells with the citric acid cycle intermediates, citrate and pyruvate, did not enhance NO-mediated (59)Fe release. Significantly, preincubation with the GSH-depleting agents, l-buthionine-[S,R]-sulfoximine or diethyl maleate, prevented NO-mediated (59)Fe mobilization. This effect was reversed by incubating cells with N-acetyl-l-cysteine that reconstitutes GSH. These results indicate that GSH levels are essential for NO-mediated (59)Fe efflux. Hence, d-Glc metabolism via the hexose monophosphate shunt resulting in the generation of GSH may be essential for NO-mediated (59)Fe release. These results have important implications for intracellular signaling by NO and also NO-mediated cytotoxicity of activated macrophages that is due, in part, to iron release from tumor target cells. (+info)
Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III).
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Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition. (+info)
Regulation of endothelial heme oxygenase activity during hypoxia is dependent on chelatable iron.
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The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia. (+info)
Protection against oxidant-mediated lysosomal rupture: a new anti-apoptotic activity of Bcl-2?
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Bcl-2 antagonizes apoptosis through mechanisms which are not completely understood. We have proposed that apoptosis is initiated by minor lysosomal destabilization followed some time later by secondary massive lysosomal rupture. In J774 cells over-expressing Bcl-2, early oxidant-induced lysosomal destabilization is unaffected but secondary lysosomal rupture and apoptosis are suppressed, despite the fact that wild-type and Bcl-2 over-expressing cells degrade hydrogen peroxide at similar rates. It may be that Bcl-2 directly blocks the effects of released lysosomal enzymes and/or prevents downstream activation of unknown cytosolic pro-enzymes by released lysosomal hydrolases, suggesting a new and heretofore unknown activity of Bcl-2. (+info)
Protection from ototoxicity of intraperitoneal gentamicin in guinea pig.
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BACKGROUND: Aminoglycoside antibiotics are common to treat peritonitis and exit-site infections in patients on peritoneal dialysis. Ototoxicity (loss of hearing or balance) is a well-documented adverse effect of aminoglycosides, and severe ototoxic reactions have been noted in patients receiving these drugs by intraperitoneal lavage. We have proposed a free-radical hypothesis for the mechanism of aminoglycoside ototoxicity and suggested a therapeutic prevention by the concomitant administration of antioxidants or iron chelators. Here we investigate whether 2, 3-dihydroxybenzoate can prevent the ototoxicity of intraperitoneal gentamicin. METHODS: Two strains of pigmented guinea pigs received daily intraperitoneal injections of gentamicin. Both strains developed ototoxicity, although different dosages were needed to produce similar auditory deficits (120 mg gentamicin base/kg body weight daily for 19 days vs. 135 mg/kg for 14 days). Dihydroxybenzoate was administered intraperitoneally once or twice daily. Auditory thresholds were measured by evoked brain stem response. Pathology was assessed as a loss of sensory cells in surface preparations of the organ of Corti. RESULTS: The auditory threshold shifts and hair cell loss were similar to the pathology observed following subcutaneous injections of gentamicin. Animals sustained almost complete loss of outer hair cells in the basal cochlea and a progressive hearing loss with threshold shifts of 60 dB at 18 kHz. The concomitant administration of dihydroxybenzoate significantly attenuated the threshold shift to less than 30 dB and reduced the loss of hair cells. The treatment with dihydroxybenzoate did not affect serum gentamicin levels. CONCLUSIONS: Antioxidant therapy is a promising approach to prevent aminoglycoside-induced hearing loss following intraperitoneal application. (+info)