Lysosomal Iron,Iron Chelation,and Cell Death

Abstract

Significance: Lysosomes are acidic organelles containing more than fifty hydrolases that provide for the degradation of intracellular and endocytosed materials by autophagy and heterophagy, respectively. They digest a variety of macromolecules, as well as all organelles, and their integrity is crucial. As a result of the degradation of iron-containing macromolecules (e.g., ferritin and mitochondrial components) or endocytosed erythrocytes (by macrophages), lysosomes can accumulate large amounts of iron. This iron occurs often as Fe(II) due to the acidic and reducing lysosomal environment. Fe(II) is known to catalyze Fenton reactions, yielding extremely reactive hydroxyl radicals that may jeopardize lysosomal membrane integrity during oxidative stress. This results in the release of hydrolases and redox-active iron into the cytosol with ensuing damage or cell death. Lysosomes play key roles not only in apoptosis and necrosis but also in neurodegeneration, aging, and atherosclerosis. Recent Advances: The damaging effect of intralysosomal iron can be hampered by endogenous or exogenous iron chelators that enter the lysosomal compartment by membrane permeation, endocytosis, or autophagy. Critical Issues: Cellular sensitivity to oxidative stress is enhanced by lysosomal redox-active iron or by lysosomal-targeted copper chelators binding copper (from degradation of copper-containing macromolecules) in redox-active complexes. Probably due to higher copper levels, lysosomes of malignant cells may be specifically sensitized by such chelators. Future Directions: By increasing lysosomal redox-active iron or exposing cells to lysosomal-targeted copper chelators, it should be possible to enhance the sensitivity of cancer cells to radiation-induced oxidative stress or treatment with cytostatics that induce such stress. Antioxid. Redox Signal. 00, 000–000.

Introduction

L ysosomes are acidic vacuolar organelles whose main function is to degrade intra- and extracellular materials in processes called autophagy and heterophagy (phagocytosis), respectively. By degrading damaged and worn-out biomolecules and organelles, autophagy provides for their turnover, being critically important for cellular homeostasis (19). Autophagy can also be responsible for the degradation of cellular constituents during programmed cell death (PCD) (22). On the other hand, heterophagy is mainly the function of specialized cells, phagocytes (e.g., macrophages), which degrade other damaged cells or their parts, as well as provide protection from microorganisms (49). In unicellular organisms, such as protozoa, heterophagy is also essential for cellular nutrition (21). Besides auto- and heterophagy, lysosomes are responsible for a number of other important functions. One of the most notable of them is the regulation of apoptosis, or PCD (38) (Fig. 1).

FIG. 1.

Schematic illustration of lysosomal activities. (A) Materials are delivered to lysosomes via autophagy and heterophagy and are then degraded by lytic enzymes. The degradation products relocate to the cytosol and are used for the synthesis of new biomolecules and organelles. Damaged lysosomes can also induce cell death. (B) If lysosomes rupture, their content of lytic enzymes and low mass iron is released into the cytosol, where they attack cellular components and cause oxidative stress by the production of hydroxyl radicals. Depending on the magnitude of damage, reparative autophagy, apoptosis, or necrosis will follow. The persistent normal influx of some hydrogen peroxide causes the formation of lipofuscin in lysosomes of long-lived postmitotic cells by the peroxidation of material under degradation.

Apart from this, lysosomes function in repair after, for example, ionizing irradiation or oxidative stress. This so-called reparative autophagocytosis removes damaged cell components and organelles (8, 36). Under starvation conditions, lysosomes may help cells survive by digesting some of the cell's own components to provide building blocks for the synthesis of important macromolecules (19). In some cell types, lysosomes, or their content, can be exocytosed (49). It has been described that tumor cells use this mechanism to secrete lysosomal proteases in order to infiltrate surrounding tissue (62).

When metalloproteins are turned over, metals (such as iron, zinc, and copper) are released within lysosomes (42, 58). Iron is liberated, in particular, due to the degradation of cytochromes, hemoglobin and myoglobin, catalase and other enzymes. Moreover, lysosomal degradation is believed to be the principal mechanism of iron release from ferritin, the main cellular iron-binding protein (4, 56). As a result, lysosomes contain much higher quantities of iron than other organelles and are probably the only place where iron exists in a low-mass redox-active form (42) (Fig. 2). The amount of intralysosomal iron has a tendency to increase with age due to the incapability of cells to release iron (24).

FIG. 2.

Demonstration of lysosomal iron in HeLa cells using the sensitive cytochemical sulfide silver method. Glutaraldehyde-fixed specimens are exposed to ammonium sulfide at pH∼12 and then developed in a colloid-protected (gum arabic) solution containing silver lactate and the reducing agent hydroquinone. Tiny silver particles precipitate and gradually enlarge to a size visible by light microscopy. The process is akin to the physical development of a photographic plate. After a short development time of 25 min (A), only very iron-rich lysosomes are visible (arrows). These lysosomes most probably correspond to autophagolysosomes that are engaged in the degradation of iron-containing material, such as ferritin or mitochondrial complexes. After 40 min of development (B), a strong general lysosomal pattern is seen, reflecting the fact that most lysosomes contain some low-mass iron. [Reproduced with permission from (81)]

When cells divide, many new biological structures are synthesized, while damaged cellular components are distributed to the daughter cells and thereby diluted. This decreases the demand on autophagy and is consistent with the low degree of autophagy found in mitotically active cells (26). In contrast, nondividing cells, such as differentiated neurons and cardiac myocytes, require intensive autophagy for their homeostasis (79).

There is abundant evidence that insufficient autophagic activity may contribute to malignant transformation by promoting increased oxidative stress, genomic instability, and accumulation of damaged organelles (67). Despite active proliferation, transformed cells are usually characterized by increased autophagy and generally up-regulated lysosomal activity. This allows them to cope with degenerative changes due to altered metabolism and disturbed circulation (38, 50). In addition, they secrete increased amounts of lysosomal proteases, promoting angiogenesis as well as tumor growth and invasion (38). Based on these properties of tumor cells, autophagic inhibition can be used as an anticancer strategy (72). The destruction of cancer cells can, for instance, be stimulated by the induction of lysosome-mediated cell death mechanisms through agents that promote metal toxicity (32, 48). This review is focused on the role of lysosomal iron and, to some degree, on copper in cell death and on possible anticancer strategies.

Lysosomal Degradation Function

Lysosomes contain numerous acid hydrolases that are capable of degrading a variety of biological molecules, including proteins, lipids, carbohydrates, and nucleic acids (55). The acidic pH of the lysosomes (between 4.5 and 5.0) is provided by the function of vacuolar ATPase, an enzyme that uses ATP for pumping protons into the lysosomal lumen (51). Extracellular material enters cells for heterophagy by invagination of the plasma membrane, resulting in the formation of early endosomes (endocytosis). Intracellular substances can be delivered to lysosomes in three different ways, corresponding to three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (72). The principal lysosomal degradation pathways in mammalian cells are presented in Figure 3.

FIG. 3.

Schematic representation of endocytosis and the different autophagic pathways. Extracellular material is taken up by the cell via endocytosis by invagination of the plasma membrane and formation of an early endosome that matures into a late endosome. Lysosomal enzymes, tagged with mannose-6-phosphate, are bound to mannose-6-phosphate receptors in clathrin-coated vesicles, transported from the trans-Golgi network (TGN), and released in late endosomes that mature to lysosomes. In macroautophagy, cytosolic organelles are surrounded by a phagophore, and the structure develops into an autophagosome that may fuse with a lysosome or a late endosome, forming an autolysosome. During microautophagy, small portions of the cytosol are delivered to lysosomes via invagination of the lysosomal membrane. Proteins with a KFERQ sequence are bound by molecular chaperones, such as heat shock cognate protein of 70 kDa (HSC70), and delivered to lysosomes via chaperone-mediated autophagy. In long-lived postmitotic cells, the undegradable pigment lipofuscin accumulates in lysosomes as a result of peroxidation of intralysosomal material destined for degradation. Lysosomes that for the time being are not active in degradation are called resting lysosomes. LE, lysosomal enzymes.

In macroautophagy, portions of cytoplasm that can contain organelles, such as mitochondria, are sequestered by membrane cisterns (phagophores) that form autophagosomes, which are double-membrane-bounded vacuoles. Neither early endosomes nor autophagosomes are acidic. They are also devoid of lysosomal enzymes. If they fuse with each other, amphisomes are formed (86). Early endosomes gradually progress to late endosomes and mature lysosomes. This process is associated with increasing endosome acidification and the transport of lysosomal enzymes by small vesicles that pinch off from the trans-Golgi network (TGN). Lysosomal enzymes are delivered in mannose-6-phosphate receptor (MPR)-bound form, suggesting that late endosomes as well as Golgi-derived vesicles and TGN contain MPR. The acidic pH triggers the release of acid hydrolases into the endosomes, while MPR are recycled back to the TGN (49). Mature lysosomes differ from late endosomes by a lower pH, a higher concentration of acid hydrolases, and the absence of MPR, indicating that the transport of the lysosomal enzymes is completed. It is suggested that autophagosomes and amphisomes can also receive acid hydrolases from Golgi-derived vesicles (82). Alternatively, autophagosomes and amphisomes receive lysosomal enzymes by fusion with late endosomes or mature lysosomes, suggesting that autophagic and heterophagic degradation pathways work together (86).

Molecular mechanisms of macroautophagy have been extensively investigated through analyses of yeast mutants with affected vacuolar degradation (53). More than thirty proteins (Atg proteins) and corresponding genes (ATG-genes) involved in macroautophagy in yeast, as well as their mammalian orthologs, have been identified (87). These proteins are responsible for the initiation of autophagy, formation and expansion of the phagophore, closure and completion of the autophagosome, and its docking and fusion with endosomes and/or lysosomes. A detailed description of molecular processes involved in macroautophagy is reviewed elsewhere (52, 72, 87). In some cases, macroautophagy can occur in the absence of certain Atg proteins (so-called noncanonical autophagy) (18).

In microautophagy, portions of the cytoplasm enter endosomes or lysosomes through invagination of the limiting membrane, often resulting in the formation of multiple intralysosomal vesicles. This pathway provides for the degradation of soluble macromolecules and small organelles (72). Although a number of proteins involved in microautophagy have been identified in yeast (46), the molecular machinery of this process in mammals remains unknown. It may be an automatic process, as lysosomes in a percoll solution spontaneously show the invagination of a microautophagocytotic type (1).

CMA, described in higher eukaryotes, is a selective process. Specific proteins with a particular pentapeptide motif KFERQ (for lysine-phenylalanine-glutamate-arginine-glutamine) are recognized by a heat shock cognate protein of 70 kDa (HSC70). The complex then binds at the lysosomal outer surface to the lysosome-associated membrane protein type 2A (LAMP-2A) (72). On binding, the substrate is transported across the lysosomal membrane.

Autophagy is an ongoing self-renewal process that occurs at a basic level under normal conditions but increases dramatically due to starvation, hypoxia, oxidative stress, ionizing radiation, and other forms of damage (40, 76). The regulation of macroautophagy has been more extensively studied and is better understood than that of microautophagy and CMA. Transcription factor EB (TFEB) and mammalian target of rapamycin complex 1 (mTORC1) are believed to play a central role in the regulation of macroautophagy and lysosomal biogenesis in mammals (69, 71). Under normal conditions, mTORC1 is located at the lysosomal surface and phosphorylates TFEB, preventing its translocation to the nucleus. Under starvation and other stress conditions, mTORC1 is detached from the lysosomes and inactivated. As a result, TFEB is not phosphorylated and, therefore, translocates to the nucleus, triggering the expression of multiple genes that are responsible for lysosomal biogenesis and autophagy [described in detail in (71)].

The bulk of cellular degradative activity is performed by lysosomes. However, many proteins, mainly short-lived ones, are degraded by multicatalytic proteinase complexes, proteasomes (57), and calpains. The latter are calcium-dependent neutral proteases that are active in the cytosol and mitochondria (35, 73). Mitochondrial proteins can also be degraded by matrix Lon and membrane-bound AAA proteases (3, 6).

Lysosomes and Iron Metabolism

Iron circulating in the bloodstream is mostly transferrin-bound Fe(III) that enters cells via receptor-mediated endocytosis. Due to acidification of iron-containing endosomes (called siderosomes), iron is released, while transferrin and its receptor are recycled back to the plasma membrane. Fe(III) is then reduced to Fe(II) by ferrireductase and transported to the cytosol by the divalent metal transporter 1 (DMT1) (56). There are also indications that such endosomal iron can be directly transferred to mitochondria through a “kiss and run” mechanism (65).

Cytosolic iron is mainly stored in nonredox-active form as a component of ferritin (i.e., iron combined with apoferritin), which possesses ferroxidase activity, transforming Fe(II) to Fe(III) (10, 56). When iron is needed for synthetic processes, it is released from ferritin. There is evidence that this occurs intralysosomally through the autophagic degradation of ferritin (29, 37, 45). Due to the acidic pH of lysosomes and the presence of reducing agents such as cysteine and glutathione, Fe(III) is converted to the redox-active Fe(II) (42, 70). Another reducing agent inside lysosomes contributing to the formation of Fe(II) may be the superoxide anion radical (O₂ ^•–) that can form in lysosomes from mitochondria undergoing autophagic degradation. Conceivably, the production of superoxide can even increase in such mitochondria due to the disabling of the electron transport complexes by lysosomal proteases (78). Fe(II) can then be relocated to the cytoplasm, probably by DMT1 or mucolipin 1 (23, 42). Some iron may be released from ferritin after its degradation by proteasomes (20, 89). It has also been suggested that iron can be directly released through pores in ferritin molecules (47, 74), although the evidence for this mechanism is weak (Fig. 4).

FIG. 4.

The role of lysosomes in cellular iron metabolism. Transferrin binds iron in the serum and is taken up by cells as a transferrin-iron complex via receptor-mediated endocytosis. At a pH of around 6 in late endosomes, iron is released from transferrin and transported to the cytosol, where it may be bound by low-affinity chelators that constitute the labile iron pool (it is unclear whether such iron is redox active or not), while the transferrin receptor is recycled back to the membrane to take up more iron. Iron is then quickly stored in ferritin or used for the synthesis of iron-containing proteins. When iron-saturated ferritin, other iron containing proteins, or organelles are degraded inside lysosomes, redox-active iron is temporarily released that makes the lysosomal membrane susceptible to peroxidation and subsequent rupture under oxidative stress. If so, lysosomal enzymes would be released to the cytoplasm and redox-active iron would relocate and cause further damage under conditions of continued oxidative stress. In contrast, when apoferritin or partly iron-loaded ferritin (or other proteins with iron-binding capacity) are autophagocytosed, they can temporarily bind redox-active iron. If there is a constant influx of such proteins by autophagy, the redox-active iron level of lysosomes is kept low and lysosomal membrane damage is avoided.

Apart from ferritin, lysosomes degrade a number of other iron-containing macromolecules, including cytochromes and several enzymes (42). In cardiac myocytes and skeletal muscle fibers, a large part of lysosomal iron is derived from autophagocytosed myoglobin, while macrophages receive iron from heterophagocytosed erythrocytes (56). Due to the continuous degradation of ferruginous materials, lysosomes are particularly rich in low mass iron, which is mainly present in its redox-active Fe(II) form (see above). This makes lysosomes particularly sensitive to oxidative stress as compared with other organelles (42). In iron overload, such as in hemochromatosis, lysosomes accumulate considerable amounts of iron, mainly in the form of hemosiderin. This brown insoluble pigment is basically derived from denatured, incompletely degraded ferritin and contains FeO in its core (63).

Even under normal conditions, proteins (including iron-containing ones) are not completely degraded. This is the result of a continuously ongoing oxidative damage to macromolecules under autophagic degradation. As a consequence, long-lived postmitotic cells, such as cardiac myocytes and neurons, progressively accumulate lipofuscin (age pigment), an intralysosomal indigestible, autofluorescent material (13, 81). Lipofuscin has been shown to contain high amounts of iron and consists of protein residues that are linked by aldehyde bridges (34). Lipofuscin-rich cells show increased sensitivity to oxidative stress, probably due to the high content of iron within lipofuscin-loaded lysosomes (75). Most importantly, progressive lipofuscin accumulation decreases the lysosomal degradative capacity of aged cells (77). As a result, aged postmitotic cells cannot effectively recycle their damaged mitochondria and, consequently, die because of lack of ATP. This deteriorative mechanism is described in detail in the mitochondrial-lysosomal axis theory of aging (14, 81).

When iron-binding proteins, such as apoferritin, ferritin with remaining iron-binding capacity, metallothioneins, and heat shock protein of 70 kDa (HSP70), enter lysosomes for recycling, they can temporarily bind redox-active iron before being degraded. Thereby, they decrease oxidant-induced lysosomal membrane permeabilization (LMP) and consequent apoptosis (42) (Fig. 5). This may explain why certain malignant cells, being rich in iron-binding stress proteins, show resistance to ionizing radiation and antineoplastic drugs (9, 60). In contrast, when ferritin is highly saturated (e.g., in hemochromatosis), the lysosomal compartment is enriched with iron, and cells are highly sensitive to oxidative injury (42).

FIG. 5.

Effects of lysosomal iron. (A) A high concentration of lysosomal redox-active iron resulting from the degradation of iron-containing metalloproteins sensitizes lysosomes to oxidative stress, and the influx of only moderate amounts of hydrogen peroxide may then cause lysosomal rupture due to the production of highly reactive hydroxyl radicals that peroxidize the lysosomal membrane. (B) In contrast, in lysosomes that autophagocytose proteins with iron-binding capacity these proteins can, before their degradation, temporarily bind iron. If the influx of such proteins is continuous, the level of lysosomal-free redox-active iron is kept low. Therefore, little HO• is produced that is absorbed by material under degradation, forming small amounts of lipofuscin in long-lived postmitotic cells. In damaged cells (e.g., after virus infections or irradiation) with a high degree of reparative autophagy, lipofuscin (often named ceroid) is also formed in cells other than long-lived postmitotic ones.

Lysosomal Iron and Oxidative Stress

Normal biologic respiration is associated with continuous electron leak from the mitochondrial electron transport chain, resulting in the continuous formation of hydrogen peroxide that easily passes biological membranes. In addition, hydrogen peroxide is produced by various oxidoreductases that operate by one-electron transfers. Although catalase and glutathione peroxidase effectively degrade most hydrogen peroxide, some of it may diffuse into lysosomes before being degraded [reviewed in (80)].

As mentioned earlier, lysosomal iron is present mainly in its Fe(II) form, being able to catalyze the Fenton reaction, that is, a homolytic splitting of hydrogen peroxide yielding the extremely reactive hydroxyl radical: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}\begin{align*}{ \rm Fe^{2 + } + H_2 O_2 \rightarrow Fe^{3 + } + HO^{ \bullet} + OH^{ - }}\end{align*}\end{document}

Hydroxyl radicals (with the half life of around 10⁻⁹ s) attack biomolecules undergoing autophagic degradation, causing their oxidative modification. Under normal conditions, with a relatively low, steady-state autophagic activity and low production of hydrogen peroxide, this results in a gradual accumulation of lipofuscin pigment within long-lived postmitotic cells (see above and Fig. 5). In enhanced autophagy (e.g., associated with the repair after cellular injury by hypoxia, ionizing radiation or various toxic agents, or when lysosomal degradation is inhibited), a more rapid formation of the pigment occurs. This pigment, which is similar to lipofuscin by the composition and mode of origin, is often called ceroid (13).

Lysosomes experience pronounced oxidative injury during enhanced oxidative stress, when cells are exposed to relatively high doses of oxidants, or in iron overload. This results in damage to their membranes and increased LMP. As a consequence, lysosomal enzymes are released to the cytosol, triggering apoptosis (see below). Lysosomes within the same cell show a wide variation in their sensitivity to oxidative stress. In cell culture models, the stability of lysosomes can be assessed by their uptake of acridine orange or other acidotropic fluorochromes (80) (Fig. 6). It was demonstrated that while some lysosomes can be damaged by a relatively slight oxidative stress, others remain stable even after exposure to severe oxidative stress (58). Such a variation in lysosomal vulnerability by oxidative stress can depend, in particular, on the different iron content of lysosomes (Fig. 2). Apparently, cells survive when only a few lysosomes are damaged, while the apoptotic machinery is activated when the injury is more pronounced (2).

FIG. 6.

Evaluation of lysosomal stability using acridine orange (AO). AO is a lysosomotropic, metachromatic weak base (pKa=10.3) that, therefore, becomes charged (AOH⁺) in acidic organelles and is retained by proton trapping in lysosomes (pH 4.5–5.5). On excitation with blue light, AO dimers (high AO concentration) exhibit red and monomers (low AO concentration) green fluorescence. Excitation with green light only shows the red fluorescence of highly concentrated AO. (A) The AO release technique can be used to follow early lysosomal damage in cells. For this purpose, cultured cells are exposed in the dark to 10 μg/ml AO for 15 min in a standard culture medium at 37°C, during which time AO accumulates as AOH⁺ in lysosomes. Thereafter, cells are rinsed with culture medium in the dark and incubated under standard culture conditions for another 10 min to reduce cytosolic AO. After the exposure of cells to damaging conditions, lysosomal membrane permeability (LMP) may be detected using flow cytofluorometry or fluorescence microscopy. Ruptured lysosomes release AO into the cytoplasm that can be observed by increased green cytoplasmic fluorescence compared with control cells when exposed to blue excitation light. Since AO is also a photosensitizer, illumination during microscopy needs to be kept at a minimum. (B) The end stage of lysosomal damage may be evaluated by the AO uptake method. Here, cells are exposed for 15 min to 10 μg/ml AO in a standard culture medium, as described earlier, after lysosomal damage. Since AO will only accumulate in intact lysosomes, the extent of lysosomal rupture can be assessed by comparing the amount of red fluorescence (using green excitation light) in these cells with the red fluorescence in control cells that are not subjected to lysosomal damage.

Lysosomes and PCD

Apoptosis, or PCD, is an evolutionarily conserved mechanism that is used for the removal of redundant, damaged, or diseased cells (28, 30). Apoptosis is essential for the elimination of cells undergoing malignant transformation, as well as for tissue remodeling during development, that is associated with the periodic removal of certain cell groups (28, 30). During apoptosis, cells undergo a controlled enzymatic digestion and are phagocytosed by their neighbors or macrophages. This prevents excessive damage of surrounding tissues and consequent inflammation. A release of toxic substances from dying cells results in necrosis (85).

It is now well established that both caspases and autophagy can be responsible for the degradation of cellular constituents during apoptosis (28, 30). Morphologically, apoptosis is characterized by chromatin condensation and fragmentation of the nucleus and cytoplasm into apoptotic bodies. In some cases, multiple autophagic vacuoles form as well. This mode of PCD is sometimes called autophagic cell death (22). The distinction between classical (caspase-dependent) apoptosis and autophagic cell death is, however, obscure, as the processes involved in the two types of cell death are often combined and even work in concert (28, 30). Moreover, even in classical apoptosis, lysosomal enzymes significantly contribute to cell death (see below), suggesting the indirect involvement of autophagy in the process.

Caspases are a family of cytosolic cysteine endopeptidases that are activated by two pathways (33). The extrinsic apoptotic pathway is initiated after the stimulation of death receptors, such as tumor necrosis factor receptors, on the plasma membrane. The intrinsic apoptotic pathway is triggered in response to internal cell injury, which is mediated by mitochondrial depolarization and the release of cytochrome c, second mitochondria-derived activator of caspases/direct IAP binding protein with low pI (SMAC/DIABLO), and some other regulatory proteins. Signaling from death receptors leads to caspase-8 activation, while the mitochondrial pathway results in the activation of caspase-9. Caspases 8 and 9, called initiation caspases, activate execution caspases such as 3 and 7, which are responsible for the final cleavage of cellular constituents [extensively reviewed in (28, 30, 38)]. Both receptor-mediated and mitochondrial pathways may be interdependent and converge at the execution stage (28, 30) (Fig. 7).

FIG. 7.

Involvement of lysosomes in apoptotic pathways. The extrinsic apoptotic pathway is triggered by the ligation of death receptors on the cell surface with subsequent caspase activation. Caspases can also be set off through the intrinsic apoptotic pathway by the activators released from mitochondria or lysosomes. A cross-talk between lysosomes and mitochondria seems to be involved not only early in the intrinsic apoptotic pathway, but also in the later stages of the extrinsic pathway. Cathepsins (Cat) released from lysosomes may either damage mitochondria directly or via activation of pro-apoptotic proteins, such as BH3 interacting-domain death agonist (Bid) and Bcl-2-associated X protein (Bax), while hydrogen peroxide released from mitochondria has damaging effects on lysosomes (particularly on iron-rich ones). Fas, Fas ligand; PLA2, phospholipase A2; TNFα, tumor necrosis factor α; TRAIL, TNF-related apoptosis-inducing ligand; SMAC/DIABLO, second mitochondria-derived activator of caspases/direct IAP binding protein with low pI.

When caspases are inhibited, injured cells are still able to undergo PCD due to mitochondria-to-nucleus translocation of apoptosis-inducing factor (AIF), causing DNA fragmentation and chromatin condensation (28, 30). In some cases, the progress of PCD can be disabled soon after its induction, for example, when viruses that infect cells encode anti-apoptotic proteins. Such cells develop necrosis-like death, which is called programmed (regulated) necrosis (27).

LMP, resulting in the release of acid hydrolases into the cytosol, was observed in various normal and malignant cells at early stages of apoptosis, often before any mitochondrial alterations (11, 38). Apparently, lysosomal enzymes, such as cathepsins B and D, induce mitochondrial permeability transition either directly or by activating phospholipase A2, promoting cytoplasmic translocation of cytochrome c, AIF, and other pro-apoptotic agents (90). Recent studies suggest that lysosomal enzymes can induce mitochondrial permeability transition more specifically, through the cleavage of BH3 interacting-domain death agonist (Bid) (cathepsins B, H, L, S, and K) or the activation of Bcl-2-associated X protein (Bax) (cathepsin D) (17, 38). In addition, cathepsins have been reported to directly activate caspases (7, 88). Lysosomal enzymes can also be involved in cell death that is secondary to other pro-apoptotic factors. In particular, the pro-apoptotic protein Bax, activated through death receptor signaling, has been shown to induce LMP, along with its previously known effect on the mitochondrial permeability transition (83).

Lysosome-Mediated Cell Death and Metal Chelators

There is extensive evidence that LMP often occurs secondary to oxidative stress, the degree of which greatly depends on the lysosomal content of low-mass iron (25, 44, 68). Moreover, due to preferential accumulation of excessive iron within lysosomes, LMP is apparently the main mechanism underlying cell death in situations of iron overload (42).

The role of lysosomal low-mass iron in apoptosis is consistent with numerous reports that show the protection of cells by iron chelators. Endocytosis of the potent iron chelator desferrioxamine (DFO) before the induction of apoptosis significantly increased the viability of various cell types (2, 12, 59, 91). DFO and other iron chelators have also been shown to have a radioprotective effect, suggesting that iron-catalyzed oxidation of lysosomal membranes and consequent LMP contribute to radiation-induced cell damage (43, 60). This is probably due to the fact that ionizing radiation triggers reparative autophagy (see above), resulting in increased lysosomal uptake of ferruginous materials and consequent accumulation of low-mass iron within lysosomes.

Apart from DFO, other iron chelators, such as the water-soluble 1-propyl-2-methyl-3-hydroxypyrid-4-one (CP22) and the lipid-soluble salicylaldehyde isonicotinoyl hydrazone (SIH), have been shown to protect cells from oxidative and radiation-induced damage. Unlike DFO, which accumulates within lysosomes leading to iron depletion, these chelators can be washed away without further negative consequences for iron metabolism (43, 54).

Neoplastic cells are characterized by increased uptake of iron, which is needed to facilitate their high proliferation rate (39). As noted earlier, they also show enhanced autophagic rate, resulting in the accumulation of large amounts of iron within the lysosomal compartment. Based on these properties of malignant cells, several anticancer strategies have been proposed. First, the inhibition of macroautophagy has been found to be effective in enhancing the toxicity of several antineoplastic drugs [reviewed in (72)]. Second, high intralysosomal iron content makes neoplastic cells increasingly sensitive to various oxidants and ionizing radiation (see above). Consistent with this, it has been found that radiation-resistant tumors contain high levels of metal-binding proteins, such as ferritin, metallothioneins, and heat shock proteins, all of which reduce the amounts of intralysosomal redox-active iron (5, 29, 41). Third, large doses of metal chelators can induce the iron starvation of malignant cells (39, 64).

Recently, a number of tridentate chelators with antineoplastic activity have been developed (64). Some of them have very low LD₅₀ values and, interestingly, are also active when applied in the form of iron complexes (sic!), creating some doubt about their activity being exclusively a result of iron chelation. The structural formulas and pKa values of several of these metal chelators indicate that they are lysosomotropic and will undergo “proton trapping” in an acidic compartment (48, 64, 66). If these tridentate chelators bind copper as well as iron, the tridentate copper chelates should be very redox active at a 1:1 chelator/copper ratio (only three out of the four copper coordinates are bound) and be able to react with a variety of reducing agents, including cysteine that is abundant in the lysosomal compartment (61). In contrast, iron is bound at a 2:1 chelator/iron ratio with all six iron coordinates bound, thereby preventing redox activity. Copper is a much more potent catalyst of Fenton-type reactions than iron. Consequently, the small amounts of hydrogen peroxide that usually reach the lysosomal compartment by diffusion might be enough to induce substantial production of hydroxyl radicals in lysosomes, especially in malignant cells that may be already under increased oxidant stress.

Many malignancies have been found to be significantly richer in copper than normal cells [reviewed in (15, 31)], and we may, thus, assume that tumor lysosomes would be rich in low-mass copper. Neocuproine and diethyl dithiocarbamate are examples of such lysosomotropic copper chelators that may promote the redox activity of copper by binding some but not all coordinates leading to LMP. The well-known tendency of tumor cells to generate excess acid may further promote the accumulation of chelators that are susceptible to proton trapping. We expect to see in the near future the initiation of clinical trials on selected copper chelators, because they have already proved to be effective in malignant cells in culture (15, 16, 31). Recently, the group of Des Richardson demonstrated a particularly efficient antineoplastic activity of the lysosomotropic copper chelator Dp44mT (48, 84).

Surprisingly, apoptosis induced by iron depletion after exposure to SIH is preceded by lysosomal destabilization, indicating that lysosomal damage can be induced not only by iron-dependent oxidative stress but also by iron depletion (44). Oxidative stress occurring simultaneously with iron starvation due to general iron chelation should clearly not induce lysosomal labilization. Therefore, the destabilizing effect on lysosomes of iron starvation should be a consequence of other apoptogenic stimuli that are yet to be identified. The finding, nevertheless, provides additional support to the emerging view that LMP is a much more common phenomenon in apoptosis than has so far been recognized. A selection of molecules and processes that damage lysosomes is depicted in Figure 8.

FIG. 8.

Examples of molecules and processes that are known to induce LMP. Lysosomes may be destabilized in a variety of ways. A high lysosomal redox-active iron content sensitizes lysosomes to oxidative stress in the form of hydrogen peroxide or ionizing irradiation (when hydrogen peroxide is produced in cells that are not anoxic). Copper chelators that are targeted to bind intralysosomal copper in a redox-active way also induce lysosomal damage via oxidative stress. On the other hand, the depletion of intralysosomal iron by certain iron chelators also leads to the rupture of lysosomes. Activated caspases and phospholipases, as well as the pro-apoptotic proteins Bax and lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains (LAPF), damage the lysosomal membrane. Examples of synthetic agents that cause lysosomal damage are o-methylserine dodecylamide hydrochloride (MSDH) and lysosomotropic aldehydes (e.g., 3-aminopropanal). The detergent sphingosine is formed during the degradation of sphingolipids by ceramidase. For further information, see the text.

Conclusion

Autophagy is a nonstop life-sustaining mechanism that provides for the efficient removal of damaged or worn-out cellular constituents, including various macromolecules, biological membranes, and organelles. Lysosomes degrade a variety of iron-containing proteins, which are delivered to them through autophagy or endocytosis, resulting in lysosomal enrichment with iron. As a potent pro-oxidant, iron promotes oxidative damage to macromolecules, making them undegradable and resulting in the progressive accumulation of intralysosomal waste material, lipofuscin, within long-lived postmitotic cells. Intralysosomal iron damages lysosomal membranes, resulting in their permeabilization and apoptosis induction. Based on this knowledge, antioxidant defense, in particular by using iron chelators, could be considered a possible anti-aging strategy.

Tumor cells are characterized by active autophagy and increased iron uptake that are needed to maintain their high proliferation rate. The use of iron chelators, leading to iron depletion, has been found to be effective in killing certain tumor cells. Similarly, copper chelators, resulting in copper chelation in a redox-active complex, have been found useful as well. Other lysosomal-targeted anti-cancer strategies involve the damage of iron-rich tumor cells by oxidants or ionizing radiation, as well as the inhibition of autophagy.

Footnotes

Acknowledgment

The authors would like to thank Professor John W. Eaton for helpful discussions and linguistic corrections.

Abbreviations Used

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