Reactive oxygen species (ROS) have been identified as signaling molecules in various pathways regulating both cell survival and cell death. Autophagy, a self-digestion process that degrades intracellular structures in response to stress, such as nutrient starvation, is also involved in both cell survival and cell death. Alterations in both ROS and autophagy regulation contribute to cancer initiation and progression, and both are targets for developing therapies to induce cell death selectively in cancer cells. Many stimuli that induce ROS generation also induce autophagy, including nutrient starvation, mitochondrial toxins, hypoxia, and oxidative stress. Some of these stimuli are under clinical investigation as cancer treatments, such as 2-methoxyestrodial and arsenic trioxide. Recently, it was demonstrated that ROS can induce autophagy through several distinct mechanisms involving Atg4, catalase, and the mitochondrial electron transport chain (mETC). This leads to both cell-survival and cell-death responses and could be selective toward cancer cells. In this review, we give an overview of the roles ROS and autophagy play in cell survival and cell death, and their importance to cancer. Furthermore, we describe how autophagy is mediated by ROS and the implications of this regulation to cancer treatments.

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O 2 KÀ ) and hydrogen peroxide (H 2 O 2 ). However, the relative functions of O 2 KÀ and H 2 O 2 in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H 2 O 2 . We found that O 2 KÀ was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O 2 KÀ and H 2 O 2 . Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O 2 KÀ levels but increased H 2 O 2 levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O 2 KÀ and H 2 O 2 levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O 2 KÀ levels, lowered H 2 O 2 levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H 2 O 2 induced autophagy and increased intracellular O 2 KÀ but failed to increase intracellular H 2 O 2 . Taken together, these findings indicate that O 2 KÀ is the major ROS-regulating autophagy.

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H 2 O 2 ) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H 2 O 2 or 2-ME-induced cell death. H 2 O 2 and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H 2 O 2 or 2-ME through overexpression of manganesesuperoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H 2 O 2 -or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H 2 O 2 or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in nontransformed cells.

Commentary 161Introduction Autophagy is a stress-induced catabolic process involving the lysosome (or, in yeast, the analogous vacuole), which is conserved in all eukaryotes (Esclatine et al., 2009; Klionsky, 2005). According to the different pathways by which cargo is delivered to the lysosome or vacuole, autophagy is divided into three main types: chaperone-mediated autophagy (CMA), microautophagy and macroautophagy (Klionsky, 2005). CMA is a process that has been characterized in higher eukaryotes but not in yeast. In CMA, a chaperone protein binds first to its cytosolic target substrate and then to a receptor on the lysosomal membrane where the unfolding of the protein occurs. The unfolded cytosolic target protein is subsequently translocated directly into the lysosome for its degradation (Massey et al., 2004). Microautophagy translocates cytoplasmic materials into the lysosome or vacuole for degradation by either direct invagination, protrusion, or septation of the lysosomal or vacuolar membrane (Wang and Klionsky, 2004). Macroautophagy is characterized by the formation of a cytosolic double-membrane vesicle, the autophagosome. During macroautophagy, cytoplasmic proteins, organelles or other materials are surrounded by phagophores, which expand and close to form autophagosomes. These autophagosomes fuse with lysosomes (or vacuoles) to form autolysosomes, in which the cytoplasmic cargos are degraded by resident hydrolases. The resulting degradation products are then transported back into the cytosol through the activity of membrane permeases for reuse (Fig. 1). In the yeast Saccharomyces cerevisiae, microautophagy engulfs cytosolic materials through an autophagic tube, which then scissions within the vacuole to release the contents into a vesicle within the vacuolar lumen for degradation (Uttenweiler and Mayer, 2008); microautophagy-like processes, such as one type of selective peroxisome degradation, are slightly different and involve targeted sequestration of the cargo (Dunn et al., 2005). The process and mechanism of microautophagy in mammalian cells are still not clear (Cuervo, 2004). Among the three main forms of autophagy, macroautophagy is the most widely studied and best characterized process. In this review, we will thus focus on macroautophagy, hereafter referred to as autophagy.Although autophagy is generally considered to be nonspecific, there are many examples of selective autophagy, including mitophagy (for mitochondria), ribophagy (for ribosomes), pexophagy (for peroxisomes) and reticulophagy (for the endoplasmic reticulum, ER) (He and Klionsky, 2009). By contrast, the yeast cytoplasm-to-vacuole targeting (Cvt) pathway is a biosynthetic pathway used to transport the vacuolar hydrolases -mannosidase and aminopeptidase I (Ape1) from the cytosol to the vacuole under normal growth conditions. As the Cvt pathway shares the core autophagy machinery, which is composed of 17 autophagy-related (Atg) proteins found in all autophagy pathways, it is also defined as a type of selective autophagy (Inoue...

Ferroptosis is an iron-dependent, oxidative cell death, and is distinct from apoptosis, necrosis and autophagy. In this study, we demonstrated that lysosome disrupting agent, siramesine and a tyrosine kinase inhibitor, lapatinib synergistically induced cell death and reactive oxygen species (ROS) in MDA MB 231, MCF-7, ZR-75 and SKBr3 breast cancer cells over a 24 h time course. Furthermore, the iron chelator deferoxamine (DFO) significantly reduced cytosolic ROS and cell death following treatment with siramesine and lapatinib. Furthermore, we determined that FeCl3 levels were elevated in cells treated with siramesine and lapatinib indicating an iron-dependent cell death, ferroptosis. To confirm this, we treated cells with a potent inhibitor of ferroptosis, ferrastatin-1 that effectively inhibited cell death following siramesine and lapatinib treatment. The increase levels of iron could be due to changes in iron transport. We found that the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. Knocking down of transferrin resulted in decreased cell death and ROS after treatment. In addition, ferroportin-1 (FPN) is an iron transport protein, responsible for removal of iron from cells. We found its expression is decreased after treatment with siramesine alone or in combination with lapatinib. Overexpression FPN resulted in decreased ROS and cell death whereas knockdown of FPN increased cell death after siramesine and lapatinib treatment. This indicates a novel induction of ferroptosis through altered iron regulation by treating breast cancer cells with a lysosome disruptor and a tyrosine kinase inhibitor.