Proteomic analysis of enriched lysosomes at early phase of camptothecin-induced apoptosis in human U-937 cells

A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-δ). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.     

alpha-tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilisation

Alpha-Tocopheryl succinate (alpha-TOS), but not a-tocopherol, triggered apoptosis in Jurkat T cells. Apoptosis was induced by alpha-TOS in a time- and concentration-dependent mode, and signs of apoptosis were visible at concentrations of alpha-TOS as low as 30 microM, and within 3-5 h after addition of the ester. Employing a specific fluorogenic substrate, caspase-3 was found to be activated rapidly in response to alpha-TOS at 50 microM. We also found that Jurkat T cells challenged with alpha-TOS, when exposed to the lysosomotropic weak base acridine orange, showed decreased lysosomal uptake of the dye. This is suggestive of the involvement of lysosomal destabilisation in apoptosis of the cells. Apoptosis of Jurkat T cells induced with alpha-TOS also involved a drop in the mitochondrial membrane potential, although this phenomenon occurred after the initiation of lysosomal rupture. All apoptotic features observed with alpha-TOS were very similar to those found when cross-linking of the Fas receptor triggered apoptosis. These findings are consistent with the recent idea that vitamin E can contribute to elimination of malignant cells by the induction of apoptosis, and can be of (patho)physiological significance.     

Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.     

Pre-clinical antitumour evaluation of Biphosphinic Palladacycle Complex in human leukaemia cells

Previous studies reported by our group have introduced a new antitumoural drug called Biphosphinic Palladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60 and Jurkat), but not in normal human lymphocytes. IC₅₀ values obtained for both cell lines using the MTT and trypan blue exclusion assays 5 h after BPC treatment were lower than 8.0 μM. Using metachromatic fluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Furthermore, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatin condensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lysosomal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation, which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We conclude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptotic signalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no toxic effects for human lymphocytes were observed, we suggest that BPC is more selective for transformed cells, mainly due to their exacerbated lysosome expression.     

Lysosomal labilization

The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.     

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)_n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes and DNA fragmentation. In this study we examined how lysosomal damage causes DNA fragmentation in LeuLeuOMe-treated HL-60 cells. When acridine orange was employed to monitor lysosomal membrane integrity, orange or red granular fluorescence was seen in normal cells. In contrast, LeuLeuOMe-treated cells showed orange, yellow or green cellular fluorescence all over the cytoplasm, suggesting that LeuLeuOMe induced a loss of lysosomal membrane integrity. The loss was inhibited by a DPPI inhibitor, GlyPheCHN₂ (GFCHN₂), but not by a caspase-3 inhibitor, Ac-DEVD-CHO, indicating that a condensation product was responsible for the loss. LeuLeuOMe also induced the activation of caspase-3-like protease and DNA fragmentation, both of which were inhibited by either GFCHN₂ or Ac-DEVD-CHO. Therefore, the activation of caspase-3-like protease links the loss of lysosomal membrane integrity to DNA fragmentation during apoptosis induced by LeuLeuOMe.     

Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant-mediated AT II cell apoptosis using an in vitro model of H(2)O(2)-stress. H(2)O(2) stress led to time-dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad-spectrum caspase inhibitor z-VAD-fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death.     

Apoptosis supercedes necrosis in mitochondrial DNA-depleted Jurkat cells by cleavage of receptor-interacting protein and inhibition of lysosomal cathepsin

In the present study, we used mitochondrial DNA-depleted Jurkat subclones (rho0 cells) to demonstrate that Fas agonistic Ab (CH-11), at the concentrations that evoke apoptotic death of the parental Jurkat cells, induced necrosis mainly through generation of excess reactive oxygen species, lysosomal rupture, and sequential activation of cathepsins B and D, and in minor part through activation of receptor-interacting protein (RIP). In the rho0 cells treated with CH-11, ATP supplementation converted necrosis into apoptosis by the formation of the apoptosome and subsequent activation of procaspase-3. In these ATP-supplemented rho0 cells (ATP-rho0), generation of excess ROS and lysosomal rupture were still seen, yet cathepsins B and D were inactivated and RIP was degraded. The conversion of necrosis to apoptosis, RIP degradation, and cathepsin inactivation in ATP- rho0 cells were blocked by caspase-3 inhibitors. Activities of cathepsins B and D in the lysate of necrotic rho0 cells were inhibited by the addition of apoptotic parental Jurkat cell lysate. Thus, apoptosis may supercede necrosis.     

Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages

Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH₄Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders.     

Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.     

Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7β-hydroxycholesterol (7βOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24 h, which was preceded by early lipid accumulation (6 h) and lysosomal membrane permeabilization (6−12 h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7βOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7βOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.     

Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells

Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3.     

Oxidative stress, growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak

Oxidative stress, growth factor starvation, and activation of the Fas/APO-1/CD95 receptor all induce apoptosis in a variety of cell-types, including the established human Jurkat T-cell line. Oxidative stress, in the form of exposure of the cells to a bolus dose of hydrogen peroxide, results in intralysosomal, iron-catalyzed oxidative reactions. This is accompanied by a time- and dose-dependent lysosomal destabilization--as evaluated by a decreased lysosomal uptake of the metachromatic fluorochrome, and weak base, acridine orange--in combination with leakage to the cytosol of lysosomal contents, including hydrolytic enzymes. Moderate lysosomal rupture is followed by apoptosis within initially intact plasma membranes, while necrosis and cell lysis are associated with a more complete lysosomal breach. Prior endocytosis of the potent iron-chelator desferrioxamine, resulting in binding of intralysosomal low molecular weight iron in a non-redox active form, largely prevents not only oxidative stress-induced lysosomal labilization, but apoptosis as well. When apoptosis is induced by the use of a monoclonal IgM anti-human Fas/APO-1/CD95 receptor antibody, the apoptotic process is again found to be accompanied by lysosomal leak. It is, however, not prevented by a preceding endocytosis of desferrioxamine and, consequently, could not be a function of intralysosomal iron-catalyzed oxidative reactions, but must be due to other mechanisms. Growth factor starvation of Jurkat cultures for a few days results in a high proportion of apoptotic cells, which contain lysosomes many of which have lost their proton gradient and appear to have released their contents. Overall, our results indicate that lysosomal leakage/rupture precedes apoptosis in Jurkat cells regardless of the initiating agent, but that such rupture may occur through multiple mechanisms. Lysosomal enzymes, leaking out of their normal vacuolar compartment, may then induce apoptosis, perhaps by proteolytic activation of the caspase-family of enzymes. Regardless of the precise mechanism, these observations suggest that partial rupture of the acidic vacuolar compartment may be one of the final pathways in apoptosis.     

Lysosomal and mitochondrial pathways in miltefosine-induced apoptosis in U937 cells

Hexadecylphosphocholine (HePC) is an anticancer agent whose effect has been shown to involve apoptosis induction but the signaling pathways leading to apoptosis remain to be elucidated. We show here that HePC induces activation of caspase-9, -3, and -8 via the intrinsic pathway, release of cytochrome c, activation and relocation of Bax to the mitochondria as well as the cleavage of Bid. Moreover, a lysosomal pathway characterized by partial lysosomal rupture, cathepsin B activation and relocation from lysosomes to the cytosol, is involved in HePC-induced apoptosis. A cathepsin B/L inhibitor partially suppresses caspase activation and apoptosis induction, indicating signaling between lysosomes and mitochondria. Conversely, the pancaspase inhibitor Q-VD-OPH inhibits lysosomal rupture, but only at early time points, suggesting that immediate lysosomal rupture involves caspases. Overexpression of Bcl-2, an anti-apoptotic protein known to prevent mitochondrial dysfunction, totally abrogates lysosomal destabilization and cell death.     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

Oxidative stress,growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak

Abstract

Oxidative stress, growth factor starvation, and activation of the Fas/APO-1/CD95 receptor all induce apoptosis in a variety of cell-types, including the established human Jurkat T-cell line. Oxidative stress, in the form of exposure of the cells to a bolus dose of hydrogen peroxide, results in intra-lysosomal, iron-catalyzed oxidative reactions. This is accompanied by a time- and dose-dependent lysosomal destabilization — as evaluated by a decreased lysosomal uptake of the metachromatic fluorochrome, and weak base, acridine orange —in combination with leakage to the cytosol of lysosomal contents, including hydrolytic enzymes. Moderate lysosomal rupture is followed by apoptosis within initially intact plasma membranes, while necrosis and cell lysis are associated with a more complete lysosomal breach. Prior endocytosis of the potent iron-chelator desferrioxamine,resulting in binding of intralysosomal low molecular weight iron in a non-redox active form, largely prevents not only oxidative stress-induced lysosomal labilization, but apoptosis as well. When apoptosis is induced by the use of a monoclonal IgM anti-human Fas/APO-1/CD95 receptor antibody, the apoptotic process is again found to be accompanied by lysosomal leak. It is, however, not prevented by a preceding endocytosis of desferrioxamine and, consequently, could not be a function of intralysosomal iron-catalyzed oxidative reactions,but must be due to other mechanisms. Growth factor starvation of Jurkat cultures for a few days results in a high proportion of apoptotic cells, which contain lysosomes many of which have lost their proton gradient and appear to have released their contents. Overall, our results indicate that lysosomal leakage/rupture precedes apoptosis in Jurkat cells regardless of the initiating agent, but that such rupture may occur through multiple mechanisms. Lysosomal enzymes, leaking out of their normal vacuolar compartment, may then induce apoptosis, perhaps by proteolytic activation of the caspase-family of enzymes. Regardless of the precise mechanism, these observations suggest that partial rupture of the acidic vacuolar compartment may be one of the finalpathways in apoptosis.     

Lysosomal destabilization contributes to apoptosis of germinal center B-lymphocytes.

During germinal center (GC) reactions, B-lymphocytes with high-affinity B-cell receptors are selected. Regulation of apoptosis is a key process in selecting such wanted B-cells and in eliminating B-cells with unwanted specificities. In this paper, we show that apoptosis in human GC B-cells involves lysosomal destabilization, which is strictly controlled by caspase-8 activity, but not by caspase-9 activity. Ligation of CD40 provides resistance to lysosomal destabilization. Experimental lysosomal rupture by the lysosomotropic drug O-methyl-l-serine dodecylamide hydrochloride (MSDH) induces apoptosis in GC B-cells, including phosphatidyl serine exposure, mitochondrial inactivation, and DNA fragmentation. These apoptotic features occur in the absence of caspase-3 activity. Follicular dendritic cells (FDCs) protect binding B-lymphocytes from lysosomal destabilization, in both the absence and the presence of MSDH. Our study demonstrates that lysosomal leakage induces apoptosis of GC B-cells in a caspase-3-independent manner and that high-affinity binding to FDCsprevents lysosomal leakage and apoptosis in GC B-cells.     

Intralysosomal iron chelation protects against oxidative stress-induced cellular damage

Oxidant-induced cell damage may be initiated by peroxidative injury to lysosomal membranes, catalyzed by intralysosomal low mass iron that appears to comprise a major part of cellular redox-active iron. Resulting relocation of lytic enzymes and low mass iron would result in secondary harm to various cellular constituents. In an effort to further clarify this still controversial issue, we tested the protective effects of two potent iron chelators--the hydrophilic desferrioxamine (dfo) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (sih), using cultured lysosome-rich macrophage-like J774 cells as targets. dfo slowly enters cells via endocytosis, while the lipophilic sih rapidly distributes throughout the cell. Following dfo treatment, long-term survival of cells cannot be investigated because dfo by itself, by remaining inside the lysosomal compartment, induces apoptosis that probably is due to iron starvation, while sih has no lasting toxic effects if the exposure time is limited. Following preincubation with 1 mM dfo for 3 h or 10 microM sih for a few minutes, both agents provided strong protection against an ensuing approximately LD50 oxidant challenge by preventing lysosomal rupture, ensuing loss of mitochondrial membrane potential, and apoptotic/necrotic cell death. It appears that once significant lysosomal rupture has occurred, the cell is irreversibly committed to death. The results lend strength to the concept that lysosomal membranes, normally exposed to redox-active iron in high concentrations, are initial targets of oxidant damage and support the idea that chelators selectively targeted to the lysosomal compartment may have therapeutic utility in diminishing oxidant-mediated cell injury.     

Oxidative stress induces macroautophagy of amyloid β-protein and ensuing apoptosis

There is increasing evidence for the toxicity of intracellular amyloid β-protein (Aβ) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Aβ in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Aβ that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Aβ-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Aβ content (Vector < APPwt < APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.     

Autophagy, ageing and apoptosis: the role of oxidative stress and lysosomal iron

As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells--probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term.