Relocalized redox-active lysosomal iron is an important mediator of oxidative-stress-induced DNA damage.
ABSTRACT: Oxidative damage to nuclear DNA is known to involve site-specific Fenton-type chemistry catalysed by redox-active iron or copper in the immediate vicinity of DNA. However, the presence of transition metals in the nucleus has not been shown convincingly. Recently, it was proposed that a major part of the cellular pool of loose iron is confined within the acidic vacuolar compartment [Yu, Persson, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1243-1252; Persson, Yu, Tirosh, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1295-1305]. Consequently, rupture of secondary lysosomes, as well as subsequent relocation of labile iron to the nucleus, could be an important intermediary step in the generation of oxidative damage to DNA. To test this concept we employed the potent iron chelator DFO (desferrioxamine) conjugated with starch to form an HMM-DFO (high-molecular-mass DFO complex). The HMM-DFO complex will enter cells only via fluid-phase endocytosis and remain within the acidic vacuolar compartment, thereby chelating redox-active iron exclusively inside the endosomal/lysosomal compartment. Both free DFO and HMM-DFO equally protected lysosomal-membrane integrity against H2O2-induced oxidative disruption. More importantly, both forms of DFO prevented H2O2-induced strand breaks in nuclear DNA, including telomeres. To exclude the possibility that lysosomal hydrolases, rather than iron, caused the observed DNA damage, limited lysosomal rupture was induced using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride; subsequently, hardly any DNA damage was found. These observations suggest that rapid oxidative damage to cellular DNA is minimal in the absence of redox-active iron and that oxidant-mediated DNA damage, observed in normal cells, is mainly derived from intralysosomal iron translocated to the nucleus after lysosomal rupture.
Project description:Jurkat cells in culture were exposed to oxidative stress in the form of continuously generated hydrogen peroxide, obtained by the addition of glucose oxidase to the medium. This treatment induced a rapid, dose-dependent increase in the ICIP (intracellular calcein-chelatable iron pool). Early destabilization of lysosomal membranes and subsequent nuclear DNA strand breaks were also observed, as evaluated by the Acridine Orange relocation test and the comet assay respectively. Somewhat later, these effects were followed by a lowered mitochondrial membrane potential, with release of cytochrome c and apoptosis-inducing factor. These events were all prevented if cells were pretreated with the potent iron chelator DFO (desferrioxamine) for a period of time (2-3 h) long enough to allow the drug to reach the lysosomal compartment following fluid-phase endocytosis. The hydrophilic calcein, a cleavage product of calcein acetoxymethyl ester following the action of cytosolic esterases, obviously does not penetrate intact lysosomal membranes, thus explaining why ICIP increased dramatically following lysosomal rupture. The rapid decrease in ICIP after addition of DFO to the medium suggests draining of cytosolic iron to the medium, rather than penetration of DFO through the plasma membrane. Most importantly, these observations directly connect oxidative stress and resultant DNA damage with lysosomal rupture and the release of redox-active iron into the cytosol and, apparently, the nucleus.
Project description:The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions.
Project description:Deferoxamine (DFO) is a high-affinity Fe (III) chelator produced by Streptomyces pilosus. DFO is used clinically to remove iron from patients with iron overload disorders. Orally administered DFO cannot be absorbed, and therefore it must be injected. Here we show that DFO induces ferritin degradation in lysosomes through induction of autophagy. DFO-treated cells show cytosolic accumulation of LC3B, a critical protein involved in autophagosomal-lysosomal degradation. Treatment of cells with the oral iron chelators deferriprone and desferasirox did not show accumulation of LC3B, and degradation of ferritin occurred through the proteasome. Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, resulted in degradation of ferritin by the proteasome. These results indicate that ferritin degradation occurs by 2 routes: a DFO-induced entry of ferritin into lysosomes and a cytosolic route in which iron is extracted from ferritin before degradation by the proteasome.
Project description:Osteosarcoma cellular iron concentration is higher than that in normal bone cells and other cell types. High levels of cellular iron help catalyze the Fenton reaction to produce reactive oxygen species (ROS), which promotes cancer cell proliferation. Dihydroartemisinin (DHA), a classic anti-malarial drug, kills plasmodium through iron-dependent ROS generation. In this research, we observed the anti-osteosarcoma effects and mechanisms of DHA. We found that DHA induced ROS production, caused mitochondrial damage, and activated autophagy via stimulation of the ROS/Erk1/2 pathway. As the storage site for a pool of ferrous iron, lysosomes are often the key organelles affected by drugs targeting iron. In this study, we observed that DHA induced lysosomal superoxide production, leading lysosomal membrane permeabilization (LMP), and autophagic flux blockage. By reducing or increasing cellular iron using deferoxamine (DFO) or ferric ammonium citrate (FAC), respectively, we found that DHA inhibited osteosarcoma in an iron-dependent manner. Therefore, iron may be a potential adjuvant for DHA in osteosarcoma treatment.
Project description:Desferrioxamine (DFO) is a bacterial siderophore with a high affinity for iron, but low cell penetration. As part of our ongoing project focused on DFO-conjugates, we synthesized, purified, characterized and studied new mtDFOs (DFO conjugated to the Mitochondria Penetrating Peptides TAT49-57, 1A, SS02 and SS20) using a succinic linker. These new conjugates retained their strong iron binding ability and antioxidant capacity. They were relatively non toxic to A2780 cells (IC50 40-100 ?M) and had good mitochondrial localization (Rr +0.45 -+0.68) as observed when labeled with carboxy-tetramethylrhodamine (TAMRA) In general, mtDFO caused only modest levels of mitochondrial DNA (mtDNA) damage. DFO-SS02 retained the antioxidant ability of the parent peptide, shown by the inhibition of mitochondrial superoxide formation. None of the compounds displayed cell cycle arrest or enhanced apoptosis. Taken together, these results indicate that mtDFO could be promising compounds for amelioration of the disease symptoms of iron overload in mitochondria.
Project description:To investigate the role of intralysosomal redox-active iron in oxidative stress-induced damage in trabecular meshwork (TM) cells.Chronic oxidative stress was applied using the hyperoxic model; acute oxidative stress was applied with H(2)O(2). Microarray analysis was performed using microarrays. mRNA and protein levels were quantified by real-time PCR and Western blot analysis, respectively. Redox-active iron was monitored using calcein-AM. Apoptosis was quantified using double staining. DNA damage was evaluated by single-cell gel electrophoresis assay. Lysosomal permeabilization was monitored using uptake and acridine orange relocation techniques. Intracellular ROS production was quantified using H(2)DCFDA. Cytosolic translocation of cathepsins was visualized with pepstatin-A-BODIPY-FL. Chemical inhibition of cathepsins was achieved with leupeptin and pepstatin A. Silencing of cathepsin expression was accomplished with miRNA sequences. Lysosomal iron chelation was achieved with desferrioxamine.Chronically stressed TM cells showed elevated levels of redox-active iron and altered expression of genes involved in intracellular iron homeostasis. Although iron increased ROS production and lipofuscin levels and sensitized TM cells to H(2)O(2), intralysosomal iron chelation completely protected the cells against H(2)O(2)-induced cell death and apoptosis. The protective effect of desferrioxamine was mediated by the prevention of lysosomal ROS generation and the rupture of lysosomal membrane, with the subsequent release of cathepsin D into the cytosol.These results indicate that the generation of intralysosomal ROS induces lysosomal membrane permeabilization and the release of cathepsin D into the cytosol, leading to TM cell death. Here, the authors propose a mechanism by which oxidative stress might contribute to the decrease in cellularity reported in the TM tissue with both aging and disease.
Project description:Chelation therapy is frequently used to help reduce excess iron in the body, but current chelators such as deferoxamine (DFO) are plagued by short blood circulation times, which necessitates infusions and can cause undesirable toxic side effects in patients. To address these issues, polyrotaxanes (PR) were synthesized by threading ?-cyclodextrin (?-CD) onto poly(ethylene glycol) bis(amine) (PEG-BA, MW 3400 g/mol) capped with enzymatically cleavable bulky Z-L phenylalanine (Z-L Phe) moieties. The resulting PR was conjugated to DFO and hydroxypropylated to generate the final polyrotaxane-DFO (hPR-DFO). The iron chelating capability of hPR-DFO was verified by UV-vis absorption spectroscopy and the ability of materials to degrade into smaller CD-conjugated DFO fragments (hCD-DFO) in the presence of the protease was confirmed via gel permeation chromatography. In vitro studies in iron-overloaded macrophages reveal that hPR-DFO can significantly reduce the cytotoxicity of the drug while maintaining its chelation efficacy, and that it is more rapidly endocytosed and trafficked to lysosomes of iron-overloaded cells in comparison to non-iron-overloaded macrophages. In vivo studies indicate that iron-overloaded mice treated with hPR-DFO displayed lower serum ferritin levels (a measure of iron burden in the body) and could eliminate excess iron by both the renal and fecal routes. Moreover, there was no gross evidence of acute toxicological damage to the liver or spleen.
Project description:Mouse erythrocytes were incubated with oxidizing agents, phenylhydrazine, divicine and isouramil. With all the oxidants a rapid release of iron in a desferrioxamine (DFO)-chelatable form was seen and it was accompanied by methaemoglobin formation. If the erythrocytes were depleted of GSH by a short preincubation with diethyl maleate, the release of iron was accompanied by lipid peroxidation and, subsequently, haemolysis. GSH depletion by itself did not induce iron release, methaemoglobin formation, lipid peroxidation or haemolysis. Rather, the fate of the cell in which iron is released depended on the intracellular availability of GSH. In addition, iron release was higher in depleted cells than in native ones, suggesting a role for GSH in preventing iron release when oxidative stress is imposed by the oxidants. Iron release preceded lipid peroxidation. The latter was prevented when the erythrocytes were preloaded with DFO in such a way (preincubation with 10 mM-DFO) that the intracellular concentration was equivalent to that of the released iron, but not when the intracellular DFO was lower (preincubation with 0.1 mM-DFO). Extracellular DFO did not affect lipid peroxidation and haemolysis, suggesting again that the observed events occur intracellularly (intracellular chelation of released iron). The relevance of iron release from iron complexes in the mechanisms of cellular damage induced by oxidative stress is discussed.
Project description:We have re-examined the lysosomal hypothesis of oxidative-stress-induced apoptosis using a new technique for exposing cells in culture to a low steady-state concentration of H(2)O(2). This steady-state technique mimics the situation in vivo better than the bolus-administration method. A key aspect of H(2)O(2)-induced apoptosis is that the apoptosis is evident only after several hours, although cells may become committed within a few minutes of exposure to this particular reactive oxygen species. In the present work, we were able to show, for the first time, several correlative links between the triggering effect of H(2)O(2) and the later onset of apoptosis: (i) a short (15 min) exposure to H(2)O(2) caused almost immediate, albeit limited, lysosomal rupture; (ii) early lysosomal damage, and later apoptosis, showed a similar dose-related response to H(2)O(2); (iii) both events were inhibited by pre-treatment with iron chelators, including desferrioxamine. This compound is known to be taken up by endocytosis only and thus to become localized in the lysosomal compartment. After exposure to oxidative stress, when cells were again in standard culture conditions, a time-dependent continuous increase in lysosomal rupture was observed, resulting in a considerably lowered number of intact lysosomes in apoptotic cells, whereas non-apoptotic cells from the same batch of oxidative-stress-exposed cells showed mainly intact lysosomes. Taken together, our results reinforce earlier findings and strongly suggest that lysosomal rupture is an early upstream initiating event, and a consequence of intralysosomal iron-catalysed oxidative processes, when apoptosis is induced by oxidative stress.
Project description:DNA damage signaling pathways are initiated in response to chemical reagents and radiation damage, as well as in response to hypoxia. It is implicated that structural maintenance of chromosomes 1 (SMC1) is not only a component of the cohesion complex but also facilitates the activation of DNA damage checkpoint proteins. Here, we studied the mechanism of DNA damage checkpoint activated by ATR-SMC1 pathway when cells are treated with desferrioxamine (DFO), a hypoxia-mimetic reagent. We show that DFO treatment induces phosphorylation of SMC1 at Ser966, NBS1 at Ser343, Chk1 at Ser317, Chk2 at Thr68, and p53 at Ser15. Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs. DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells. Expression of SMC1 S966A in which Ser966 is substituted to Ala attenuates apoptosis and phosphorylation of Chk1 at Ser317 after DFO treatment, although levels of HIF1? are not significantly changed. These results suggest that DFO induces apoptosis through the ATR-SMC1 arm of the pathway.