Autophagy supports survival and phototransduction protein levels in rod photoreceptors

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomes for degradation. Defective autophagy can contribute to the age-dependent accumulation of damaged proteins and organelles leading to altered cellular homeostasis and loss of function.^{1, 2, 3, 4, 5} The metabolic roles of autophagy can be classified into two types, basal and induced. In nutrient-rich conditions, autophagy is suppressed but still occurs at low levels (basal autophagy); however, when cells are subjected to stress (starvation, injury, hypoxia), autophagy is activated immediately (induced autophagy).⁶ Induced autophagy maintains the amino acid pool inside cells to adapt to starvation while constitutive autophagy has been shown to function as a cell-repair mechanism that is important for long-lived postmitotic cells.^{7, 8, 9, 10, 11} Defects in autophagy have been associated with neurodegenerative diseases,^{12, 13, 14, 15} diabetes,^{16, 17} lysosomal storage disease¹⁸ and the loss of vision.¹⁹ In addition to macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) have been described. Although little is known about microautophagy in mammalian cells, macroautophagy (hereafter autophagy) is a major pathway for bulk degradation of cytoplasmic components. CMA is a more selective pathway for degradation of cytosolic proteins that can compensate for the loss of macroautophagy.^{2, 20, 21, 22}Inherited retinal degenerative diseases such as retinitis pigmentosa or Leber''s congenital amaurosis are characterized by premature and progressive death of rod and cone photoreceptor cells.²³ These diseases are characterized by the loss of night vision due to the death of rods followed by the loss of cones leading to diminished visual acuity and a reduction in the quality of life for patients. Disease is typically associated with the production of harmful gene products that promote pathology by inhibiting critical pathways resulting in cell death.^{24, 25, 26} Strategies to prevent photoreceptor death during retinal degenerative disease such as gene replacement therapies or inhibition of cell death pathways have been undertaken with some success;^{27, 28, 29} however, effective treatments for these blinding disorders are lacking.Another strategy that could be used in conjunction with other therapies might be to enhance survival by stimulating autophagy. Augmenting autophagy would increase the supply of nutrients to stressed cells and accelerate removal of damaged proteins thereby prolonging cell survival beyond what would be possible by only preventing cell death. Although canonical^{22, 30, 31, 32, 33} and noncanonical autophagic mechanisms³⁴ have been detected in the eye, our knowledge of basic autophagy functions in this organ is still limited. In order to understand how autophagy maintains retinal homeostasis and function, we undertook studies to examine the consequences of deleting the essential autophagy gene Atg5 in rod photoreceptors.     

Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death

Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis.In the United States, spinal cord injury (SCI) has an annual incidence of 11 000 and prevalence of nearly 500 000. Neuronal cell death is an important contributor to SCI-induced neurological deficits. Many of the affected neurons do not die because of direct mechanical damage but rather show delayed cell death as a result of injury-induced biochemical changes (secondary injury).^{1, 2, 3, 4} Thus, blocking or attenuating secondary neuronal death may serve to limit posttraumatic disabilities.Macroautophagy (hereafter called autophagy) is a lysosome-dependent catabolic pathway degrading cytoplasmic proteins, protein aggregates and organelles.^{5, 6, 7} Autophagy is initiated by the formation of autophagosomes, double membrane vesicles containing cytoplasmic components that include potentially toxic protein aggregates and damaged organelles. Autophagosomes then fuse with lysosomes to allow degradation of their contents by lysosomal hydrolases.^{8, 9, 10, 11} This progress of cargo, from sequestration in autophagosomes, to their delivery and degradation in lysosomes, is termed autophagy flux. Autophagy flux is important for homeostasis in all cells but appears especially critical in terminally differentiated cells such as neurons.^{12, 13} It is also upregulated, and often plays a protective function, in response to cell injury.^{14, 15} For example, autophagy is activated in response to and can limit effects of homeostasis perturbation in the endoplasmic reticulum (ER stress).^{16, 17} Thus, autophagy plays an important neuroprotective function, while impaired autophagy flux has been implicated in neurodegenerative disorders such as Parkinson''s and Alzheimer''s diseases.^{18, 19, 20, 21}Upregulation of autophagy markers has been observed after SCI,^{22, 23} but its mechanisms and function remain controversial, with both beneficial and detrimental roles proposed. Under certain circumstances, pathologically increased autophagy can contribute to cell death,^{21, 24} particularly when autophagy flux is blocked, for example, because of lysosomal defects. Defects in autophagy flux can also exacerbate ER stress and potentiate ER-stress-induced apoptosis.^{16, 17} ER stress has long been implicated as part of the secondary injury after central nervous system trauma,^{25, 26} but its mechanisms remain unknown.In the current study, we characterized the temporal distribution and cell-type specificity of autophagy following contusive SCI in a rat model. Our data demonstrate that autophagosome accumulation after SCI is not due to increased initiation of autophagy, but rather due to inhibition of autophagy flux. This likely reflects the disruption of lysosomal function after SCI. Pathological accumulation of autophagosomes is prominent in ventral horn (VH) motor neurons, where it is associated with signs of ER stress and related apoptosis. Together, our findings suggest that autophagy is disrupted after SCI and may exacerbate ER stress and neuronal cell death.     

Implication of different domains of the Leishmania major metacaspase in cell death and autophagy

Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine–cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H₂O₂, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.Apoptosis is, in most cases, associated with and depends on the activation of cys-dependent peptidases, named caspases.^{1, 2} Once activated, initiator caspases induce a proteolytic cascade via the activation of effector caspases that ultimately cleave numerous substrates, thereby causing the typical morphological features of apoptosis.^{3, 4} Despite their essential role in apoptosis, caspases are also involved in non-apoptotic events, including inflammation, cell proliferation, cell differentiation⁵ and the cell survival process autophagy, a major catabolic process in eukaryotic cells that allows cells to survive nutrient starvation due to engulfment of a portion of the cytoplasm by a specific membrane, delivery to lysosomes or vacuoles and digestion by hydrolytic enzymes.^{6, 7, 8, 9, 10} Plants, fungi and protozoa are devoid of caspases but express metacaspases (MCAs).¹¹MCAs are cysteine peptidases of the clan CD, family 14, with a caspase-like histidine–cysteine catalytic dyad.^{12, 13} However, besides their distant similarity to caspases,¹⁴ MCAs prefer arginine/lysine in the P1 position, whereas caspases prefer aspartic residues.^{15, 16} The role of MCAs in cell death is still enigmatic. For example, in the yeast Saccharomyces cerevisiae, YCA1 has a role in cell death,^{17, 18} whereas, although only partly dependent on its conserved catalytic cysteine, it also facilitates the removal of unfolded proteins, prolonging cellular life span.¹⁹ Similarly, some metacaspases have roles, outside of death, in stress acclimation pathways, as in Aspergillus fumigatus²⁰ or in the unicellular planctonic organisms diatoms.^{21, 22} In Arabidopsis thaliana, AtMC1 is a positive regulator of cell death and a survival factor for aging plants,²³ whereas AtMC2 negatively regulates cell death.²⁴ Trypanosoma brucei TbMCA2, TbMCA3 and TbMCA5 and Leishmania major MCA are involved in cell cycle regulation.^{25, 26}Leishmania are parasitic protozoa responsible for the neglected tropical disease leishmaniasis, transmitted to humans by the bite of the sand fly. In the insect, parasites proliferate as free-living flagellated forms called procyclic promastigotes within the midgut before differentiating into virulent metacyclic promastigotes and migrating to the proboscis.^{27, 28} In the mammalian host, promastigotes are taken up by macrophages and transform into amastigotes. Under a variety of stress stimuli, apoptosis-like morphological and biochemical features have been described in Leishmania, among which are cell shrinkage, chromatin condensation, DNA fragmentation or mitochondrial depolarization.^{29, 30, 31, 32, 33, 34, 35, 36, 37, 38} Despite the evidence of morphological and biochemical markers of cell death in dying Leishmania, very little is known about the cell death pathway and the implicated executioner proteins. Indeed, essential proteins involved in mammalian apoptosis, death receptors, small pro- and anti-apoptotic molecules and caspases, are apparently not encoded in the genome of Leishmania³⁹ and the role of Leishmania MCA in cell death is still controversial, certain authors suggesting a role as a negative regulator of intracellular amastigote proliferation, instead of having a caspase-like role in the execution of cell death.⁴⁰LmjMCA contains different domains: an N-terminal domain with a Mitochondrion Localization Signal (MLS),⁴¹ a caspase-like catalytic domain and a C-terminal proline-rich domain.⁴¹ On the basis of this domain structure, LmjMCA can be classified among the type I metacaspases,¹⁶ a subclass more generally defined in higher plants and characterized by the presence of an N-terminal prodomain and a short linker between the large and small subunits, as initiator caspases in metazoans.¹¹ Upon induction of cell death by heat shock, H₂O₂ or drugs like miltefosine or curcumin, LmjMCA is processed and the catalytic domain is released,⁴¹ liberating the C-terminal domain. It was therefore interesting to investigate the functional roles of the different domains.In this report, we studied the role of L. major MCA (LmjMCA), using an MCA-deficient strain and overexpressing independently the catalytic and the C-terminal domains. The results confirmed that MCA was not essential to L. major survival. In contrast, LmjMCA processing, releasing its catalytic and C-terminal domains, induced cell death in L. major, whereas the overexpression of Lmjmca gene triggered autophagy after interaction of the C-terminal domain with itself and with other proteins, acting on or upstream of the autophagic protein ATG8.     

Pdcd4 deficiency enhances macrophage lipoautophagy and attenuates foam cell formation and atherosclerosis in mice

Macrophage foam cells, a major component of the atherosclerotic lesion, have vital roles in the development of atherosclerosis. Lipoautophagy, a type of autophagy characterized by selective delivery of lipid droplet for lysosomal degradation, may impact atherosclerosis by regulating macrophage foam cell formation. Previously, we reported that programmed cell death 4 (PDCD4), a tumor suppressor, negatively regulated autophagy in tumor cells. However, its roles in macrophage lipoautophagy, foam cell formation and atherosclerosis remain to be established. Here we found that Pdcd4 deficiency clearly improved oxidized low-density lipoproteins-impaired autophagy efflux, promoted autophagy-mediated lipid breakdown in murine macrophages and thus prevented macrophage conversion into foam cells. Importantly, Pdcd4 deficiency in mice significantly upregulated macrophage autophagy in local plaques along with attenuated lipid accumulation and atherosclerotic lesions in high-fat-fed Apolipoprotein E knockout mice. Bone marrow transplantation experiment demonstrated that PDCD4-mediated autophagy in hematopoietic cells contributed to the development of atherosclerosis. These results indicate that endogenous PDCD4 promotes for macrophage foam cell formation and atherosclerosis development via inhibiting autophagy and provides new insights into atherogenesis, suggesting that promoting macrophage autophagy through downregulating PDCD4 expression may be beneficial for treating atherosclerosis.Atherosclerosis is a lipid dysfunction-derived chronic inflammatory process in large and medium arterial wall.¹ Macrophage foam cell, as a major component in the lesion of atherosclerosis, has vital role in the development of atherosclerosis. In the initial step of atherosclerotic development, circulating monocytes migrate into arterial wall via dysfunctional endothelial cells and differentiate into macrophages.^{2, 3, 4} The infiltrated macrophages ingest and digest oxidized low-density lipoprotein (ox-LDL), and then transport lipid out of vascular wall.⁵ However, macrophage with overloaded lipids stored in the form of lipid droplets (LDs) will transform into foam cells. Macrophage foam cell formation could promote the development of atherosclerosis.⁶ Thus, decreasing the formation of macrophage foam cell would be an attractive strategy to reverse plaque lipid buildup.⁷The macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved and well-controlled cellular catabolic process. During the process, cytoplasmic components are sequestered in double-membrane vesicles (which is called autophagosome) and degraded by fusion with lysosomal compartments (autophagolysosome) for recycling application.⁸ The process of autophagy is regulated by several autophagy-related genes (ATGs) encoded proteins, such as ATG5, ATG6 (also known as BECN1), ATG8 (also known as microtubule-associated protein 1 light chain 3, LC3) and ATG12. ATG5 is involved in the early stage of autophagosome formation. ATG5 is conjugated with ATG12 and ATG16L to form ATG12–ATG5–ATG16L complex, which contributes to the elongation and closure of the autophagosomes in the generation of lipidated forms of LC3 family proteins.⁹ Lipoautophagy, a type of autophagy that selectively delivers LDs for lysosomal degradation,¹⁰ regulates lipid metabolism and is involved in the process of atherosclerosis.^{11, 12, 13, 14} In advanced atherosclerosis, macrophage autophagy becomes dysfunctional. However, the basic autophagy deficiency in macrophage by specific Atg5 knockout accelerates atherosclerotic plaques in high-fat-fed ldlr^−/− mice via promoting oxidative stress, plaque necrosis¹² or inflammasome hyperactivation.¹³ More interestingly, autophagy can enhance brokendown of lipid in LD, cholesterol efflux from macrophage foam cells and further inhibit atherogenisis.¹⁴ Stent-based delivery of everolimus (mTOR inhibitor) in atherosclerotic plaques of cholesterol-fed rabbits leads to a marked reduction of macrophages via autophagic cell death.¹⁵ Therefore, regulating the level of macrophage autophagy and macrophage conversion into foam cells would be a potential target for preventing the atherosclerotic plaques formation.¹⁶Programmed cell death 4 (PDCD4), an inhibitor of protein translation, inhibits translation initiation via binding to the translation initiation factor eIF4A or translation elongation by direct or indirectly binding to the coding region of specific RNAs.^{17, 18} Accumulated evidence has demonstrated PDCD4 as a tumor suppressor.¹⁹ PDCD4 can inhibit promotion and progression of tumors, such as lung cancer,²⁰ hepatocellular carcinoma cells,²¹ colon cancer,²² ovarian cancer²³ and glioma.²⁴ In addition, it has been reported that PDCD4 is also involved in the development of inflammatory diseases.^{25, 26, 27, 28, 29, 30} For example, Pdcd4-deficient mice are resistant to experimental allergic encephalitis,²⁵ LPS-induced endotoxin shock²⁶ and type-1 diabetes.²⁷ In addition, Pdcd4-deficient mice are sensitive to LPS/D-galactosamine-induced acute liver injury.²⁸ Recently, we reported that Pdcd4 deficiency attenuated adipocyte foam cells, diet-induced obesity, obesity-associated inflammation and insulin resistance,²⁹ and increased IL-10 expression by macrophages that partly involved in atherosclerosis in hyperlipidemic mice,³⁰ suggesting that PDCD4 may be involved in the metabolism-related diseases. Furthermore, we found that PDCD4 negatively regulated autophagy by inhibiting ATG5 expression in tumor cells.³¹ However, its role in macrophage lipoautophagy and foam formation, and association with atherosclerosis remain to be investigated.In the present study, we found that Pdcd4 deficiency improved ox-LDL-impaired autophagy efflux in murine macrophage and subsequently attenuated macrophage conversion into foam cells in an autophagy-dependent manner and further attenuated the formation of atherosclerotic lesions in hyperlipidemia mice. These results indicate that PDCD4 is critical for macrophage foam cell formation in atherosclerosis development and provides new insights into atherogenesis, and potential therapeutic avenues to treat atherosclerosis-associated diseases.     

The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: functional linkage with autophagy

Autophagy is a major nutrient recycling mechanism in plants. However, its functional connection with programmed cell death (PCD) is a topic of active debate and remains not well understood. Our previous studies established the plant metacaspase AtMC1 as a positive regulator of pathogen-triggered PCD. Here, we explored the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered PCD and aging. We observed that autophagy acts as a positive regulator of pathogen-triggered PCD in a parallel pathway to AtMC1. In addition, we unveiled an additional, pro-survival homeostatic function of AtMC1 in aging plants that acts in parallel to a similar pro-survival function of autophagy. This novel pro-survival role of AtMC1 may be functionally related to its prodomain-mediated aggregate localization and potential clearance, in agreement with recent findings using the single budding yeast metacaspase YCA1. We propose a unifying model whereby autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants, when these functions are not masked by the cumulative stresses of aging, and negatively regulating senescence in older plants.An emerging theme in cell death research is that cellular processes thought to be regulated by linear signaling pathways are, in fact, complex. Autophagy, initially considered merely a nutrient recycling mechanism necessary for cellular homeostasis, was recently shown to regulate cell death, mechanistically interacting with components that control apoptosis. Deficient autophagy can result in apoptosis^{1, 2, 3} and autophagy hyper-activation can also lead to programmed cell death (PCD).⁴ In addition, the pro-survival function of autophagy is mediated by apoptosis inhibition and apoptosis mediates autophagy, although this cross-regulation is not fully understood.⁵In plants, autophagy can also have both pro-survival and pro-death functions. Autophagy-deficient plants exhibit accelerated senescence,^{6, 7, 8} starvation-induced chlorosis,^{6, 7, 9} hypersensitivity to oxidative stress¹⁰ and endoplasmic reticulum stress.¹¹ Further, autophagy-deficient plants cannot limit the spread of cell death after infection with tissue-destructive microbial infections.^{12, 13} The plant phytohormone salicylic acid (SA) mediates most of these phenotypes.⁸ Autophagy has an essential, pro-survival role in situations where there is an increasing load of damaged proteins and organelles that need to be eliminated, that is, during aging or stress. Autophagy has an opposing, pro-death role during developmentally regulated cell death^{14, 15} or during the pathogen-triggered hypersensitive response PCD (hereafter, HR) that occurs locally at the site of attempted pathogen attack.^{16, 17} The dual pro-death/pro-survival functions of plant autophagy remain a topic of active debate.Also under scrutiny are possible novel functions of caspases and caspase-like proteins as central regulators of pro-survival processes. Caspases were originally defined as executioners of PCD in animals, but increasing evidence indicates that several caspases have non-apoptotic regulatory roles in cellular differentiation, motility and in the mammalian immune system.^{18, 19, 20}Yeast, protozoa and plants do not have canonical caspases, despite the occurrence of morphologically heterogeneous PCDs.²¹ More than a decade ago, distant caspase homologs termed metacaspases were identified in these organisms using structural homology searches.²² Metacaspases were classified into type I or type II metacaspases based on the presence or absence of an N-terminal prodomain, reminiscent of the classification in animals into initiator/inflammatory or executioner caspases, respectively. Despite the architectural analogy between caspases and metacaspases, differences in their structure, function, activation and mode of action exist.^{23, 24, 25}Metacaspases mediate PCD in yeast,^{26, 27, 28, 29, 30, 31} leishmania,^{32, 33} trypanosoma³⁴ and plants.²⁴ We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically regulate HR in Arabidopsis thaliana.³⁵ Our work showed that AtMC1 is a positive regulator of HR and that this function is mediated by its catalytic activity and negatively regulated by the AtMC1 N-terminal prodomain. AtMC2 antagonizes AtMC1-mediated HR.Besides AtMC2, new examples of metacaspases with a pro-life/non-PCD role are emerging. Protozoan metacaspases are involved in cell cycle dynamics^{34, 36, 37, 38} and cell proliferation.³⁹ The yeast metacaspase Yca1 alters cell cycle dynamics⁴⁰ and interestingly, is required for clearance of insoluble protein aggregates, thus contributing to yeast fitness.⁴¹Here, we explore the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered HR and aging. Our data support a model wherein autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants and negatively regulating senescence in older plants.     

Epigallocatechin-3-gallate opposes HBV-induced incomplete autophagy by enhancing lysosomal acidification,which is unfavorable for HBV replication

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, exhibits diverse beneficial properties, including antiviral activity. Autophagy is a cellular process that is involved in the degradation of long-lived proteins and damaged organelles. Recent evidence indicates that modulation of autophagy is a potential therapeutic strategy for various viral diseases. In the present study, we investigated the effect of EGCG on hepatitis B virus (HBV) replication and the possible involvement of autophagy in this process. Our results showed that HBV induced autophagosome formation, which was required for replication of itself. However, although EGCG efficiently inhibited HBV replication, it enhanced, but not inhibited, autophagosome formation in hepatoma cells. Further study showed that HBV induced an incomplete autophagy, while EGCG, similar to starvation, was able to induce a complete autophagic process, which appeared to be unfavorable for HBV replication. Furthermore, it was found that HBV induced an incomplete autophagy by impairing lysosomal acidification, while it lost this ability in the presence of EGCG. Taken together, these data demonstrated that EGCG treatment opposed HBV-induced incomplete autophagy via enhancing lysosomal acidification, which was unfavorable for HBV replication.Macroautophagy (hereafter autophagy) is a conserved cellular process through which cytoplasmic materials are sequestered into double-membrane vacuole called autophagosomes and destined for degradation through fusion with lysosomes.^{1, 2, 3} Accumulating evidence indicates that autophagy is involved in diverse pathophysiological processes, including cancer, neurodegenerative disorders, and cardiovascular diseases.^{4, 5, 6, 7} Recent studies show that autophagy has an important role in regulating the replication of many viruses, including dengue virus, coxsackievirus B3 virus (CVB3), hepatitis C virus (HCV), and influenza virus A.^{8, 9, 10, 11, 12} Several investigations also indicate that autophagy has an important role in hepatitis B virus (HBV) replication: autophagy is induced by HBV and is required for HBV replication; however, the underlying mechanisms remains still unclear.^{13, 14, 15, 16}Green tea is the most commonly consumed beverage worldwide. In traditional Chinese medicine, green tea is considered to have beneficial properties for human health, including antitumorigenic, antioxidant, and anti-inflammatory activities.^{17, 18, 19} Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and appears to be the primary active ingredient accounting for the latter''s biological effects. In recent years, EGCG is revealed to display inhibitory effect on diverse viruses, such as human immunodeficiency virus type-1, Epstein–Barr virus (EBV), and HCV.^{20, 21, 22, 23, 24, 25} Of interest, EGCG is also found to regulate autophagy formation, although it seems to be cell-type specific.^{26, 27, 28, 29, 30} Given the potential therapeutic effect of EGCG on viral infection and its role in autophagy regulation, we investigated the effect of EGCG on HBV replication and the possible involvement of autophagy in this process.Here we showed that HBV induced an incomplete autophagy that was required for HBV replication; however, a complete autophagic process induced by EGCG appeared to be unfavorable for HBV replication. Further study showed that HBV hampered the autophagic flux by impairing lysosomal acidification, which could be opposed by the treatment of EGCG.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, Bim_EL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.The intrinsic mitochondrial apoptotic pathway is required for efficient chemotherapeutic killing of cancer cells,¹ and is initiated through BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). MOMP releases cytochrome c to activate effector caspases.² Conversely, inhibitor of apoptosis protein (IAP) family members suppress initiator and effector caspases via direct binding and E3 ligase activities.^{3, 4, 5} Consequently, MOMP-induced release of Smac (second mitochondria-derived activator of caspases) from the mitochondria, to inhibit XIAP (X-chromosome-linked IAP)-mediated caspase suppression, can be required for apoptosis.⁶Autophagy, a lysosomal degradative mechanism undergoing extensive crosstalk with cell death and survival pathways,⁷ degrades damaged mitochondria in a process termed mitophagy.^{8, 9} Damaged mitochondria are targeted by lysosomal degradation through the recruitment of autophagy receptors to the outer mitochondrial membrane (OMM),⁸ or via delivery of mitochondrial-derived vesicles (MDVs) directly to the lysosome.¹⁰ The E3 ubiquitin ligase Parkin targets and ubiquitylates mitochondria, mediating both MDV degradation¹¹ and autophagy receptor-dependent mitophagy.^{12, 13} Alternatively, Fundc1¹⁴ and atypical BH3-only Nip family proteins Bnip3 and Bnip3L/Nix localize to the OMM and act as mitophagy receptors via their LC3-interacting region (LIR).^{15, 16, 17, 18} While targeting of damaged mitochondria suggests that mitophagy may counter apoptotic mitochondria, mitophagy occurs progressively over days,^{12, 14, 16, 17, 19} a rate that is likely insufficient to alter intracellular propagation of mitochondrial apoptosis, which can occur within minutes.^{20, 21} Indeed, Bnip3- and Fundc1-induced mitophagy have no direct effect on apoptosis,^{14, 18} and we determined that Bnip3-mediated mitophagy was cytoprotective if activated before apoptosis.¹⁷ While MDV delivery of mitochondria to lysosomes operates at a higher rate, minutes to hours,¹⁰ this process is regulated by Parkin and restricted to specific mitochondrial components.¹¹ Overall, for most intrinsic apoptosis scenarios it remains unknown whether lysosomal processing of mitochondria influences their capacity to activate or enhance apoptosis.Here, we used high-resolution imaging to evaluate the behavior of apoptosis, autophagy, lysosomal and ubiquitylation pathways in response to canonical (tBid, Bim_EL, Bik, Bad) and atypical (Bnip3, Bnip3L/Nix) BH3-only protein expression. We report that, in parallel to intrinsic apoptosis signaling, canonical BH3-only proteins induce the recruitment of endolysosomal machinery, in the absence of mitophagy. We determined that mitochondrial depolarization rapidly translocates the caspase inhibitor XIAP to the mitochondria. There, XIAP actuates MOMP within all mitochondria, concomitant with ubiquitylation at the OMM and inside OMM-bound regions, and triggers ubiquitin-dependent recruitment of Rab5 and its binding partners, as well as late endosomes into the mitochondria. Consequently, in a manner dependent on lysosome- and proteasome-activities, XIAP degrades its inhibitor Smac. We propose that in response to bioenergetic stress, the functional integration between lysosomes and mitochondria, mediated by XIAP and independent of autophagy, offers a novel mechanism to modulate mitochondrial apoptosis.     

IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.Autophagy is a degradative process in eukaryotic cells that removes or turns over bulk cytoplasmic constituents through the endosomal and lysosomal fusion system (i.e., autophagosomes).^{1, 2} Autophagy is induced by stressful conditions such as starvation and pathogenic invasion.²Hypertrophic scar (HS) is a major skin fibrotic disorder caused by hypercellularity and extracellular matrix (ECM) component deposition.^{3, 4, 5} HS formation is usually recognized as the consequence of disturbed tissue repair processes and/or disrupted homeostasis in the skin after traumatic injury: HS negatively impacts on patient appearance, skeletal muscle function, and quality of life in general.^{6, 7, 8, 9} About 40–70% of surgeries and over 91% of burn injuries result in HS.¹⁰ A key feature of HS is a metabolic disorder of collagen-based ECM proteins.^{11, 12, 13} Autophagy has an important role in homeostasis of tissue structure and function.^{2, 14, 15} Skin autophagic capability is associated with HS and with the pathogenesis of many human diseases.^{16, 17, 18, 19, 20, 21, 22, 23}Existing studies suggest that cytokines are important regulators of the autophagic process in both immune and non-immune cells.^{24, 25, 26} Interleukin-10 (IL10), expressed by a variety of mammalian cell types, was first described as a cytokine-synthesis-inhibitory factor with immunosuppressive and anti-inflammatory functions.^{27, 28} IL10 has a pivotal role in wound healing^{29, 30} and is a promising therapeutic agent for scar improvement in both animal models and human cutaneous wounds.^{9, 31, 32}Fibroblasts are one of the most important effector cells responsible for HS formation.^{12, 33, 34} Thus, we were prompted to elucidate the mechanisms underlying the interactions among IL10, autophagy, and HS formation, with the aim of providing a molecular foundation for the therapeutic efficacy IL10. We used HS tissue, HS-derived fibroblasts (HSFs), and starvation-induced autophagy in HSFs as our research platform.Here, we report that IL10 inhibited autophagy by interfering with IL10R-mediated activation of IL10R-STAT3, as well as by activating the AKT-mTOR pathway. In addition, cross talk among STAT3, AKT, and mTOR and between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways collaboratively regulated starvation-induced autophagy in HSFs.     

ARHI (DIRAS3)-mediated autophagy-associated cell death enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts

Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.Autophagy has a well-defined role in cellular physiology, removing senescent organelles and catabolizing long-lived proteins.^{1, 2} Under nutrient-poor conditions, the fatty acids and amino acids produced by hydrolysis of lipids and proteins in autophagolysosomes can provide energy to sustain starving cells. Prolonged autophagy is, however, associated with caspase-independent type II programmed cell death. Although the mechanism of autophagy-associated cell death has not been adequately characterized, programmed necrosis or necroptosis has been implicated in some studies.^{3, 4}Given the ability to sustain or kill cells, the role of autophagy in cancer is complex and dependent on the context of individual studies. During oncogenesis in genetically engineered mice, reduced hemizygous expression of genes required for autophagy (BECN1, Atg4, ATG5, Atg7) can accelerate spontaneous or chemically induced tumor formation,^{5, 6} suggesting that autophagy can serve as a tumor suppressor. Other observations with established cancers suggest that autophagy can sustain metabolically challenged neoplasms, particularly in settings with inadequate vascular access.^{7, 8} Autophagy has also been shown to protect cancer cells from the lethal effects of some cytotoxic drugs.^{9, 10}Our group has found that cancer cell proliferation,^{11, 12, 13} motility,¹⁴ autophagy and tumor dormancy^{15, 16} can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), that is downregulated in 60% of ovarian cancers by multiple mechanisms,^{17, 18} associated with shortened progression-free survival.¹⁹ Ovarian cancer cell sublines have been developed with tet-inducible expression of ARHI. In cell culture, re-expression of ARHI induces autophagy and clonogenic ovarian cancer cell death within 72 h.¹⁶ In xenografts, re-expression of ARHI arrests cell growth, inhibits angiogenesis and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks of induction, dormancy is broken, vascularization occurs and xenografts grow promptly. Treatment of dormant xenografts with chloroquine (CQ), a functional inhibitor of autophagy, delays tumor outgrowth, suggesting that autophagy facilitates survival of poorly vascularized, nutrient-deprived ovarian cancer cells. The relevance of this model to human disease is supported by the recent observation that small deposits of dormant ovarian cancer found on the peritoneal surface at ‘second look'' operations following initial surgery and chemotherapy exhibit autophagy and increased expression of ARHI in >80% of cases.²⁰Ovarian cancer develops in >22 000 women each year in the United States.²¹ Over the past four decades, the 5-year survival has increased from 37% to ∼50% with optimal cytoreductive surgery and combination chemotherapy using taxane- and platinum-based regimens,^{21, 22} but long-term survival and cure stand at ∼30% for all stages, due, in large part, to the persistence and recurrence of dormant, drug-resistant ovarian cancer cells. For the past two decades, standard chemotherapy for ovarian cancer has included a combination of a platinum compound and a taxane. Carboplatin and cisplatin are alkylating agents that bind covalently to DNA producing intra- and inter-strand crosslinks that, if not repaired, induce apoptosis and cell death.^{23, 24} Our previous studies suggest that ∼20% of primary ovarian cancers exhibit punctate immunohistochemical staining for LC3, a biomarker for autophagy that decorates autophagosome membranes, whereas >80% of cancers that have survived platinum-based chemotherapy exhibit punctate LC3.²⁰ Consequently, autophagy might provide one mechanism of resistance to platinum-based therapy.In this report, we have explored mechanism(s) by which ARHI induces autophagy-associated cell death and enhances cisplatin cytotoxicity. Cisplatin has been found to trigger apoptosis by inducing caspase-3 activation and PARP cleavage in ovarian cancer cells.^{25, 26} We hypothesized that autophagy-associated cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells might still be vulnerable to platinum-based chemotherapy.     

Distinct requirements of Autophagy-related genes in programmed cell death

Although most programmed cell death (PCD) during animal development occurs by caspase-dependent apoptosis, autophagy-dependent cell death is also important in specific contexts. In previous studies, we established that PCD of the obsolete Drosophila larval midgut tissue is dependent on autophagy and can occur in the absence of the main components of the apoptotic pathway. As autophagy is primarily a survival mechanism in response to stress such as starvation, it is currently unclear if the regulation and mechanism of autophagy as a pro-death pathway is distinct to that as pro-survival. To establish the requirement of the components of the autophagy pathway during cell death, we examined the effect of systematically knocking down components of the autophagy machinery on autophagy induction and timing of midgut PCD. We found that there is a distinct requirement of the individual components of the autophagy pathway in a pro-death context. Furthermore, we show that TORC1 is upstream of autophagy induction in the midgut indicating that while the machinery may be distinct the activation may occur similarly in PCD and during starvation-induced autophagy signalling. Our data reveal that while autophagy initiation occurs similarly in different cellular contexts, there is a tissue/function-specific requirement for the components of the autophagic machinery.There is a fundamental requirement for multicellular organisms to remove excess, detrimental, obsolete and damaged cells by programmed cell death (PCD).^{1, 2} In the majority of cases caspase-dependent apoptosis is the principle pathway of PCD; however, there are other modes of cell death with important context-specific roles, such as autophagy.^{3, 4} Defects in autophagy have significant adverse consequences to normal cellular functions and contribute to the pathogenesis of numerous human diseases. This is particularly evident in cancer where depending on the context autophagy can have tumour-suppressing or -promoting roles. Given the number of clinical trials targeting autophagy in cancer therapy, it will be critically important to understand the context-specific regulation and functions of autophagy.⁵Autophagy is a highly conserved multi-step catabolic process characterised by the encapsulation of part of the cytoplasm inside a double-membrane vesicle called the autophagosome. Autophagosomes then fuse with lysosomes and the components are subsequently degraded by acidic lysosomal hydrolases.⁶ The process of autophagy can be functionally divided into four groups: (1) serine/threonine kinase Atg1 (ULK1 in mammals) complex and its regulators responsible for the induction of autophagy; (2) the class III phosphatidylinositol 3-kinase (PI3K) complex, which involves Atg6 and functions in the nucleation of the autophagosome; (3) the Atg8 and Atg12 conjugation systems, which involves several Autophagy-related (Atg) proteins essential for the expansion of autophagosome; and (4) Atg9 and its associated proteins including Atg2 and Atg18, which aids the recycling of lipid and proteins.⁷ In addition, several of the Atg proteins can function in multiple steps. For example, Atg1 interacts with proteins with different functions (e.g. Atg8, Atg18 and others), suggesting that it is not only required for initiation but also participates in the formation of autophagosomes.⁸ It is yet to be fully established if the context-specific functions of autophagy have distinct requirements for select components of the autophagy pathway.High levels of autophagy are induced in response to stress, such as nutrient deprivation, intracellular stress, high temperature, high culture density, hormones and growth factor deprivation.^{9, 10} The target of rapamycin (TOR) pathway is a central mediator in regulating the response to nutrients and growth signalling. TOR functions in two distinct complexes, with regulatory associated protein of TOR (Raptor) in TOR complex 1 (TORC1) or with rapamycin insensitive companion of TOR (Rictor) in TOR complex 2 (TORC2).^{11, 12, 13, 14, 15} Of these, TORC1 regulates autophagy; in nutrient-rich conditions, TORC1 activity inhibits the Atg1 complex preventing autophagy and cellular stress such as starvation leads to inactivation of TORC1 promoting a dramatic increase in autophagy. TORC2 can also negatively regulate autophagy via the FoxO3 complex in specific context.¹⁶Most direct in vivo evidence for a role of autophagy in cell death has emerged from studies in Drosophila.⁵ Developmentally regulated removal of the Drosophila larval midgut can occur in the absence of canonical apoptosis pathway, whereas inhibiting autophagy delays the process.^{17, 18} Also, inhibition of autophagy leads to delayed degradation of larval salivary glands in Drosophila.¹⁹ Genetic studies have shown that many of the Atg genes known to be involved in starvation-induced autophagy in the Drosophila fat body are also involved in autophagy-dependent degradation of salivary glands and midgut.^{5, 20, 21} However, systematic studies to test whether starvation-induced autophagy and autophagy required for PCD require identical components have not been carried out, and there are some observations suggesting that there may be distinctions. For example, in Atg7-null mutants autophagy is perturbed but the larval–adult midgut transition proceeds normally.²² In addition, a novel Atg7- and Atg3-independent autophagy pathway is required for cell size reduction during midgut removal.²³ Here we show that downregulation of TORC1 activity is required for induction of autophagy during midgut removal. Surprisingly, however, the requirement of part of the autophagy machinery during midgut degradation was found to be distinct to that which is required during autophagy induced by starvation. We report that Atg genes required for autophagy initiation, Atg8a and recycling are all essential for autophagy-dependent midgut removal, whereas other components of the elongation and nucleation steps are not essential.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Retinal pigment epithelial cells undergoing mitotic catastrophe are vulnerable to autophagy inhibition

The increased mitochondrial DNA damage leads to altered functional capacities of retinal pigment epithelial (RPE) cells. A previous study showed the increased autophagy in RPE cells caused by low concentrations of rotenone, a selective inhibitor of mitochondrial complex I. However, the mechanism by which autophagy regulates RPE cell death is still unclear. In the present study, we examined the mechanism underlying the regulation of RPE cell death through the inhibition of mitochondrial complex I. We report herein that rotenone induced mitotic catastrophe (MC) in RPE cells. We further observed an increased level of autophagy in the RPE cells undergoing MC (RPE-MC cells). Importantly, autophagy inhibition induced nonapoptotic cell death in RPE-MC cells. These findings indicate that autophagy has a pivotal role in the survival of RPE-MC cells. We next observed PINK1 accumulation in the mitochondrial membrane and parkin translocation into the mitochondria from the cytosol in the rotenone-treated RPE-MC cells, which indicates that increased mitophagy accompanies MC in ARPE-19 cells. Noticeably, the mitophagy also contributed to the cytoprotection of RPE-MC cells. Although there might be a significant gap in the roles of autophagy and mitophagy in the RPE cells in vivo, our in vitro study suggests that autophagy and mitophagy presumably prevent the RPE-MC cells from plunging into cell death, resulting in the prevention of RPE cell loss.Cell death is a process that is both complementary and antagonistic to cell division in order to maintain tissue homeostasis, and cell death has a pivotal role in several physiological processes and diseases.¹ The most extensively studied category, apoptosis, is characterized by the massive activation of caspases, chromatin condensation, and a reduction in cell volume. Necrosis is characterized by an increase in cell volume, the swelling of organelles, and the rupture of the plasma membrane and is largely considered an accidental, uncontrolled type of cell death.² Necroptosis is a regulated necrotic cell death that is triggered by broad caspase inhibition in the presence of death receptor ligands and is characterized by necrotic cell death morphology. Autophagy is a degradative lysosomal pathway that is characterized by the accumulation of cytoplasmic material in the vacuoles for bulk degradation by lysosomal enzymes. Although autophagy has a pivotal role in cell survival, increased autophagic activity is often associated with cell death.² Mitotic catastrophe (MC) is a type of cell death that results from a failure to undergo mitosis after DNA damage, leading to tetraploidy or endopolyploidy. Cells undergoing MC usually form large cells with multiple micronuclei.³Retinal pigment epithelial (RPE) cells form a single layer of cells adjacent to the photoreceptor outer segment (POS) of the retina, and these cells have pivotal roles in the maintenance of the POS cells. RPE cell death is a significant factor in several ocular pathological conditions, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). AMD is a progressive degeneration of the macula and is broadly classified as either dry or wet. The dry form of AMD is more common and is characterized by the presence of drusen in the macula. Mitochondrial DNA variants of respiratory complex I are associated with an increased risk of AMD.⁴ Because damage to and the death of RPEs are crucial and perhaps even triggering events in AMD,⁵ protection against RPE cell death could delay the onset of AMD. Conversely, RPE cells significantly contribute to the formation of the epiretinal membrane in PVR. Thus, the induction of RPE cell death in the epiretinal membranes could be a new approach to inhibit cellular proliferation in PVR.⁶ Most studies concerning RPE cell death in the context of these ocular pathological conditions have focused on two types of cell death, apoptosis and necrosis.Although advances have been made in the understanding of RPE cell death, there is little information concerning the role of autophagy in the RPE cell death associated with these ocular pathological conditions. Each day, RPE cells phagocytose and digest the distal parts of the POS, which are ultimately degraded in the lysosomes.^{7, 8, 9} The interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.⁹ In the RPE cells of old eyes, this physiological lysosomal load may be further increased to remove damaged material, and insufficient digestion of the damaged macromolecules and organelles by old RPE cells will lead to progressive accumulation of biological ‘garbage'', such as lipofuscin.¹⁰ Thus, abnormalities in the lysosome-dependent degradation of shed POS debris can contribute to the degeneration of RPE cells. A previous study suggested that age-related changes in autophagy may underlie the genetic susceptibility found in AMD patients and may be associated with the pathogenesis of AMD.¹⁰ However, the mechanism by which autophagy regulates RPE cell demise in AMD is still unclear. The role of autophagy in the proliferation of the RPE cells in PVR and its regulation as a therapeutic strategy for PVR have not been documented yet.Rotenone, a natural isoflavonoid produced by plants, is a selective and stoichiometric inhibitor of mitochondrial complex I.¹¹ More specifically, rotenone blocks NADH oxidation by the NADH-ubiquinone oxide reductase enzymatic complex, which results in the inhibition of mitochondrial respiration and a reduction in ATP synthesis.^{12, 13, 14} Rotenone treatment also results in the production of reactive oxygen species (ROS), eventually leading to cell death.^{15, 16} Several studies have shown that rotenone causes an accumulation of autophagic vacuoles, perhaps in response to the inhibition of mitochondrial function and the generation of oxidative stress.^{17, 18, 19} Irrespective of that activity of rotenone has been lively studied in various cells, the effect of rotenone on RPE cells has rarely been studied. A previous study using an in vitro system revealed that low concentrations of rotenone resulted in mtDNA damage in RPE cells and suggested that the increased autophagy caused by rotenone treatment in aged RPE cells could affect the formation of drusen and AMD.¹⁰ However, the mechanism by which rotenone regulates RPE cell demise remains unclear.We undertook this study to elucidate the mechanism regulating the demise of RPE cells that are damaged by mitochondrial complex I inhibition. We report herein that rotenone induces MC in RPE cells. Additionally, we show that RPE cells undergoing mitotic catastrophe (RPE-MC cells) induced by mitochondrial complex I inhibition are vulnerable to autophagy inhibition.     

Spermidine induces autophagy by inhibiting the acetyltransferase EP300

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.Macroautophagy (herein referred to as ‘autophagy'') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.¹ This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 ‘puncta''), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1).²There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity.^{3, 4, 5, 6} This applies to caloric restriction or intermediate fasting,⁷ continuous or intermittent medication of rapamycin,^{8, 9, 10} administration of the sirtuin 1-activator resveratrol,^{11, 12} external supply of the polyamine spermidine,¹³ or genetic ablation of p53.¹⁴ In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan.^{13, 14, 15, 16, 17} Moreover, direct induction of autophagy by transgenic expression of autophagy-relevant genes such as ATG5 in mice is sufficient to increase lifespan.¹⁸Recently, acetyltransferases have emerged as a potential target for the pharmaceutical induction of autophagy. Thus, depletion of the sole donor of acetyl groups, acetyl-coenzyme A (acetyl-CoA), is sufficient to reduce the acetylation of cytoplasmic and nuclear proteins coupled to the induction of autophagy.^{19, 20, 21, 22} Culture of mammalian cells in nutrient-free (NF) conditions or starvation of mice for 24 h reduced the intracellular nucleocytosolic concentrations of acetyl-CoA at the same time as autophagy was induced, and replenishment of acetyl-CoA by external sources (for instance, by providing a membrane-permeant precursor of α-ketoglutarate for anaplerotic reactions or by microinjection of acetyl-CoA) was sufficient to inhibit starvation-induced autophagy.^{19, 20, 21, 22} Beyond the inhibition of acetyltransferases by acetyl-CoA depletion, direct pharmacological inhibition of acetyltransferases might also contribute to the induction of autophagy. A close correlation between autophagy induction and deacetylation of cytoplasmic proteins was observed in a screen conceived to identify autophagy-stimulating polyphenols²³ as well as in in vivo experiments designed to explore the health-improving effects of coffee.²⁴ Spermidine turned out to be an efficient inhibitor of histone acetyltransferases in vitro¹³ and reduced the global protein acetylation levels in cultured cells.^{25, 26}Driven by these premises, we investigated the hypothesis that several health-related compounds including anacardic acid, curcumin, garcinol and spermidine might induce autophagy by inhibition of acetyltranferases. Here we report results supporting this hypothesis. Moreover, we demonstrate that one particular acetyltransferase, EP300 (E1A-binding protein p300), negatively controls autophagy and that anacardic acid, curcumin, garcinol and spermidine may induce autophagy by directly inhibiting EP300.     

The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson''s disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.Nowadays, Parkinson''s disease (PD) constitutes the main motor disorder and the second neurodegenerative disease after Alzheimer''s disease. Etiology of PD remains unknown, but both environmental and genetic factors have been implicated. Among the genes associated with PD is the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) encoding gene encoded by PARK8. Indeed, LRRK2 mutations have been described in a substantial number of idiopathic late-onset PD patients without a known family history of the disease.^{1, 2, 3}The physiologic function remains unknown. It localizes in the cytosol as well as in specific membrane subdomains, including mitochondria, autophagosomes and autolysosomes,⁴ and interacts with a whole array of proteins, including both α- and β-tubulin,^{5, 6} tau,⁷ α-synuclein⁸ and F-actin.⁹ LRRK2 gene mutations, including the most common G2019S,³ are associated with increases in toxic putative kinase activity.^{1, 10} LRRK2 overexpression is toxic to cultured cells,^{11, 12} and LRRK2 loss did not cause neurodegenerative changes (for a review see Tong and Shen¹³). However, LRRK2 transgenic mice lack obvious PD-like behavioral phenotypes.¹⁴ LRRK2-associated PD patients show degeneration of dopaminergic neurons in the substantia nigra.¹⁵ Data from our own group and others have associated mitochondrial apoptotical pathways with PD,^{16, 17, 18} and, in this context, LRRK2 mutant-mediated toxicity could be due to mitochondria-dependent apoptosis.¹⁹ There is considerable evidence for impaired mitochondrial function and morphology in both early-onset, autosomal recessive inherited PD and late-onset sporadic PD.Mitochondrial dynamics include several mechanisms, such as fission, fusion and mitophagy.^{20, 21} Altered fission/fusion dynamics might be a common pathogenic pathway of neurodegenerative diseases. It is well documented that mitochondrial dynamics constitute a relevant issue in some experimental neurodegenerative models.^{20, 22, 23, 24, 25} Mitochondrial dynamics is tightly regulated by cellular pathways including those participated by the dynamin-related protein-1 (Drp1). Drp1 mostly locates in the cytoplasm, but is stimulated after fission stimuli to migrate to the mitochondria. Once there, Drp1 forms ring-like structures, which wrap around the scission site to constrict the mitochondrial membrane resulting in mitochondrial fission.^{26, 27} Interestingly, a functional interaction between PD-associated LRRK2 and members of the dynamin GTPase superfamily has been described.²⁸Macroautophagy (hereafter referred to as autophagy) is an active cellular response, which functions in the intracellular degradation system of cellular debris such as damaged organelles. Whether autophagy promotes cell death or enhances survival is still controversial.^{29, 30} It requires the formation of autophagosomes where cellular content is to be degraded by the action of lysosomal enzymatic content. Autophagosome formation is regulated by an orderly action of >30 autophagy-related (Atg) proteins. Among them is the microtubule-associated protein 1A/1B-light chain 3 (LC3), a homolog of Apg8p, which is essential for autophagy in yeast and is associated with autophagosome membranes.³¹ Interestingly, these vesicles are mostly highly mobile in the cytoplasm.³² Wild-type and mutant LRRK2 expression has been related to autophagy.^{4, 33, 34, 35, 36} Reactive oxygen species (ROS) function as relevant second messengers after several stimuli, including mitochondrial disruption. Exacerbated ROS increases might result in overactivation of antioxidant systems and yield harmful oxidative stress. Among oxidative stress hallmarks is the accumulation of α,β-unsaturated hydroxyalkenal 4-hydroxy-2-nonenal (4-HNE), whose accumulation has been reported in PD post-mortem patient brains,^{37, 38} thus giving a significant relevance to ROS in the pathogenesis of PD.All these results indicate LRRK2 as a promising pharmacologic target in PD treatment.³⁹ Several LRRK2 inhibitor drugs have been synthetized, such as the potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide (GSK2578215A). GSK2578215A exhibits biochemical IC₅₀s of 10.9 nM against wild-type LRRK2, and possesses a high ratio of brain to plasma distribution.⁴⁰ This study provides key insights into the mechanisms downstream of LRRK2 inhibition, and spreads light onto an underexplored, yet potentially tractable therapeutic target for treating LRRK2-associated PD. We demonstrate how inhibition of this kinase results in the activation of cellular death pathways such as the mitochondrial fission machinery, and how cells reply by activating a protective autophagic response. Our results show the presence of oxidative stress hallmarks, thus pointing to a key function for ROS, placed downstream of mitochondrial fission.     

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF)

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.Osteosarcoma, a common primary bone tumor in children and adolescents, is prone to early metastasis through blood.¹ Treatment with a combination of surgery and aggressive adjuvant chemotherapy has improved the survival rate of osteosarcoma patients. The 5-year-survival rates of non-metastatic patients have reached a plateau of approximately 70%.^{2, 3} However, patients with poor responses to chemotherapeutics will undergo local recurrence and metastasis, which reduce the 5-year-survival rates to only 20% despite additional doses or drugs.^{4, 5} Drug resistance is responsible for the poor prognosis. Attenuating chemoresistance facilitates better treatment of osteosarcoma.^{6, 7} Novel treatment strategies that combine anticancer drugs with adjuvant agents could improve the antitumor effects.^{8, 9}In the 1960s, macrophage migration inhibitory factor (MIF) was identified as a pluripotent protein that modulates inflammation.¹⁰ Increasing evidence suggests that inflammation is closely related to tumorigenesis.¹¹ MIF plays a bridging role between inflammation and tumorigenesis.^{12, 13, 14} MIF triggers the activation of the MAPK and PI3K pathways by binding its membrane receptor CD74, resulting in the inhibition of cell apoptosis.¹⁵ Recently, MIF was demonstrated to be involved in cell proliferation, differentiation, angiogenesis and tumorigenesis.^{16, 17, 18} Some evidence has indicated that MIF is abundantly expressed in various cancers and is significantly associated with tumor invasion and metastasis.^{19, 20, 21} MIF has been well established to be involved in the development of glioblastoma,²² breast cancer,²³ bladder cancer²⁴ and colon cancer.^{20, 25} MIF was also upregulated in osteosarcoma.^{26, 27} The knockdown of MIF blocked osteosarcoma cell proliferation and invasion.²⁶ However, the effect of MIF on drug resistance in osteosarcoma has not yet been investigated. Wu et al. ²³ have revealed that MIF knockdown promoted chemosensitivity by inducing autophagy in breast cancer. In contrast, autophagy reportedly contributed to chemoresistance in osteosarcoma.⁶ These controversial results prompted us to confirm the role of MIF in drug resistance in osteosarcoma.In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. Both N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced reactive oxygen species (ROS)-mediated autophagy to rescue cell death. Furthermore, MIF inhibition by DSTD enhances chemosensitivity by downregulating HMGB1 in osteosarcoma cells. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.