The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation

TrkA receptor activation is a pivotal process for neuronal cell differentiation and survival. However, its overactivation or removal of its ligand NGF tends to cause the cell death. Recently, we demonstrated that TrkA overexpression induces cell death via apoptosis. In this study we also show that the TrkA-mediated cell death is associated with autophagy. TrkA-induced cells revealed an increase of GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were eventually blocked by the addition of some autophagy inhibitors such as 3-methyladenine, ammonium chloride or wortmannin. In addition, although expression of autophagy-related proteins such as LC3-II or Beclin-1 was subtly altered during the TrkA-mediated cell death, depletion of ATG5 or Beclin-1 substantially decreased cell death in TrkA-expressing cells. In particular, reactive oxygen species (ROS) were dramatically accumulated in TrkA-induced cells, and the high accumulation of ROS was released by treatment of autophagy inhibitors. Furthermore, addition of an antioxidant N-acetylcysteine promoted the survival of TrkA-expressing cells and suppressed AVO production in cells. We also showed that this ROS accumulation was closely associated with reduction of catalase expression. Taken together, TrkA overexpression causes ROS accumulation via reduced catalase expression, ultimately leading to autophagic cell death.     

Apigetrin induces extrinsic apoptosis,autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell

Apigetrin is a flavonoid glycoside phytonutrient derived from fruits and vegetables that is well known for a variety of biological activities such as antioxidant and anti-inflammatory activities. In the current study, we determined the effect of apigetrin on AGS gastric cancer cell. Apigetrin reduced cancer cell proliferation and induced G2/M phase cell cycle arrest by regulating cyclin B1, cdc25c and cdk1 protein expression in AGS cell. Apigetrin treatment caused apoptotic cell death in AGS cells, characterized by the accumulation of apoptosis portion, cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Apigetrin-treated cells increased the expression of extrinsic apoptosis pathway proteins and mRNA. However, intrinsic apoptosis pathway related proteins were not altered. In addition, AGS cells treated with apigetrin increased autophagic cell death, featured by the formation of autophagic vacuole and acidic vesicular organelles. Autophagy marker proteins, such as LC3B-II and beclin-1, were increased, and p62, an autophagy flux marker protein, was also increased by endoplasmic reticulum stress. Also, the phosphorylation of PI3K/AKT/mTOR pathway proteins and its downstream targets in apigetrin-treated AGS cells was identified to be decreased. Taken together, these data suggest that apigetrin-treated AGS cells induced G2/M phase cell cycle arrest, extrinsic apoptosis and autophagic cell death through PI3K/AKT/mTOR pathway, which can lead to the inhibition of gastric cancer development. Thus, our findings strongly indicate that apigetrin is a basic natural derived compound that could be used as a nutrient source with potential anticancer activities against gastric cancer.     

Autophagic cell death induced by TrkA receptor activation in human glioblastoma cells

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.     

Autophagy in cell survival and death

Macroautophagy hereafter referred to as autophagy is a major lysosomal catabolic pathway for macromolecules and organelles conserved in eukaryotic cells. The discovery of the molecular basis of autophagy has uncovered its importance during development, life extension and in pathologies such as cancer, certain forms of myopathies and neurodegenerative diseases. Autophagy is a cell survival mechanism during starvation that is controlled by amino acids. Starvation-induced autophagy is an anti-apoptotic mechanism. However autophagy is also an alternative to apoptosis through autophagic cell death. In many situations apoptosis and autophagy can both contribute to cell dismantlement.     

综述:细胞自噬的调控机制及其在老年性痴呆发生发展中的作用

细胞自噬是生物体内一种用于清除功能异常的细胞器、错误折叠的蛋白质、被氧化的脂类等有害大分子物质的重要途径．它的机制从低等生物酵母到高等的哺乳动物都高度保守,对维持正常的生命活动至关重要．错误折叠的蛋白质若不能被有效清除,就会造成积聚,致使神经细胞功能丧失乃至死亡,这是神经退行性疾病包括老年性痴呆(Alzheimer's disease, AD)的主要原因．本文回顾了近年来关于细胞自噬及其与老年性痴呆关系的研究进展,主要内容包括以下几点：自噬参与Aβ的产生和清除;γ分泌酶中的Presenilin 1在自噬底物降解中的作用;Tau蛋白调控自噬体转运、融合;老年性痴呆早期自噬对细胞的保护;细胞中感应营养和能量的两个关键蛋白mTOR和AMPK调控自噬及其对老年性痴呆的潜在影响机制．     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Apoptotic and autophagic responses to photodynamic therapy in 1c1c7 murine hepatoma cells

Photodynamic therapy (PDT) is a process that can induce apoptosis, autophagy or both depending on the cell phenotype. Apoptosis is a pathway to cell death while autophagy can protect from photokilling or act as a death pathway. In a previous study, we reported a cytoprotective effect of autophagy in murine leukemia cell lines where both autophagy and apoptosis occur within minutes after irradiation of photosensitized cells. In this study, we examined the effects of mitochondrial photodamage catalyzed by low (≤ 1 μM) concentrations of the photosensitizing agent termed benzoporphyrin derivative (BPD, Verteporfin) on murine hepatoma 1c1c7 cells. Apoptosis was not observed until several hours after irradiation of photosensitized cells. Autophagy was clearly cytoprotective since PDT efficacy was significantly enhanced in a knockdown sub-line (KD) in which the level of a critical autophagy protein (Atg7) was markedly reduced. This result indicates that autophagy can protect from phototoxicity even when apoptosis is substantially delayed. Much higher concentrations (≥ 10 μM) of BPD had previously been shown to inhibit autophagosome formation. Phototoxicity studies performed with 10 μM BPD and a proportionally reduced light dose were consistent with the absence of an autophagic process in wild-type (WT) cells under these conditions.     

Rapamycin Induces Apoptosis When Autophagy is Inhibited in T-47D Mammary Cells and Both Processes are Regulated by Phlda1

Autophagy is an evolutionarily conserved lysosomal degradation pathway and plays a critical role in the homeostatic process of recycling proteins and organelles. Functional relationships have been described between apoptosis and autophagy. Perturbations in the apoptotic machinery have been reported to induce autophagic cell deaths. Inhibition of autophagy in cancer cells has resulted in cell deaths that manifested hallmarks of apoptosis. However, the molecular relationships and the circumstances of which molecular pathways dictate the choice between apoptosis and autophagy are currently unknown. This study aims to identify specific gene expression of rapamycin-induced autophagy and the effects of rapamycin when the autophagy process is inhibited. In this study, we have demonstrated that rapamycin is capable of inducing autophagy in T-47D breast carcinoma cells. However, when the autophagy process was inhibited by 3-MA, the effects of rapamycin became apoptotic. The Phlda1 gene was found to be up-regulated in both autophagy and apoptosis and silencing this gene was found to reduce both activities, strongly suggests that Phlda1 mediates and positively regulates both autophagy and apoptosis pathways.     

内质网应激与自噬及其交互作用影响内皮细胞凋亡

内质网应激是普遍存在于真核细胞中的应激-防御机制。在内环境稳态遭到破坏的情况下,未折叠蛋白质反应的3条信号通路,分别通过增强蛋白质折叠能力、减少蛋白质生成和促进内质网相关蛋白质降解等途径缓解细胞内压力。同时,也通过多种分子信号机制调控细胞凋亡。自噬是一种生理性的降解机制。通过形成自噬泡并与溶酶体结合摄取并水解胞内受损细胞器和蛋白质等,清除代谢废物,维持细胞正常功能。自噬缺陷或过度激活均可导致细胞凋亡或非程序性死亡。自噬的程度和细胞内压力水平有关。内质网应激通过未折叠蛋白质反应和Ca²⁺浓度变化及其相关分子信号调控自噬。自噬又可反馈性调节内质网应激反应,二者相互作用,在内皮细胞凋亡过程中发挥重要作用。未来内质网应激和自噬可作为药物靶点为内皮相关性疾病提供诊疗策略。     

Caspases: A Molecular Switch Node in the Crosstalk between Autophagy and Apoptosis

Autophagy and apoptosis are two important catabolic processes contributing to the maintenance of cellular and tissue homeostasis. Autophagy controls the turnover of protein aggregates and damaged organelles within cells, while apoptosis is the principal mechanism by which unwanted cells are dismantled and eliminated from organisms. Despite marked differences between these two pathways, they are highly interconnected in determining the fate of cells. Intriguingly, caspases, the primary drivers of apoptotic cell death, play a critical role in mediating the complex crosstalk between autophagy and apoptosis. Pro-apoptotic signals can converge to activate caspases to execute apoptotic cell death. In addition, activated caspases can degrade autophagy proteins (i.e., Beclin-1, Atg5, and Atg7) to shut down the autophagic response. Moreover, caspases can convert pro-autophagic proteins into pro-apoptotic proteints to trigger apoptotic cell death instead. It is clear that caspases are important in both apoptosis and autophagy, thus a detailed deciphering of the role of caspases in these two processes is still required to clarify the functional relationship between them. In this article, we provide a current overview of caspases in its interplay between autophagy and apoptosis. We emphasized that defining the role of caspases in autophagy-apoptosis crosstalk will provide a framework for more precise manipulation of these two processes during cell death.     

Apoptotic and autophagic responses to photodynamic therapy in 1c1c7 murine hepatoma cells

Photodynamic therapy (PDT) is a process that can induce apoptosis, autophagy or both depending on the cell phenotype. Apoptosis is a pathway to cell death while autophagy can protect from photokilling or act as a death pathway. In a previous study, we reported a cytoprotective effect of autophagy in murine leukemia cell lines where both autophagy and apoptosis occur within minutes after irradiation of photosensitized cells. In this study, we examined the effects of mitochondrial photodamage catalyzed by low (≤1 μM) concentrations of the photosensitizing agent termed benzoporphyrin derivative (BPD, Verteporfin) on murine hepatoma 1c1c7 cells. Apoptosis was not observed until several hours after irradiation of photosensitized cells. Autophagy was clearly cytoprotective since PDT efficacy was significantly enhanced in a knockdown sub-line (KD) in which the level of a critical autophagy protein (Atg7) was markedly reduced. This result indicates that autophagy can protect from phototoxicity even when apoptosis is substantially delayed. Much higher concentrations (≥10 μM) of BPD had previously been shown to inhibit autophagosome formation. Phototoxicity studies performed with 10 μM BPD and a proportionally reduced light dose were consistent with the absence of an autophagic process in wild-type (WT) cells under these conditions.     

Endoplasmic reticulum stress triggers autophagy

Eukaryotic cells have evolved strategies to respond to stress conditions. For example, autophagy in yeast is primarily a response to the stress of nutrient limitation. Autophagy is a catabolic process for the degradation and recycling of cytosolic, long lived, or aggregated proteins and excess or defective organelles. In this study, we demonstrate a new pathway for the induction of autophagy. In the endoplasmic reticulum (ER), accumulation of misfolded proteins causes stress and activates the unfolded protein response to induce the expression of chaperones and proteins involved in the recovery process. ER stress stimulated the assembly of the pre-autophagosomal structure. In addition, autophagosome formation and transport to the vacuole were stimulated in an Atg protein-dependent manner. Finally, Atg1 kinase activity reflects both the nutritional status and autophagic state of the cell; starvation-induced autophagy results in increased Atg1 kinase activity. We found that Atg1 had high kinase activity during ER stress-induced autophagy. Together, these results indicate that ER stress can induce an autophagic response.     

Highlights from this issue

Autophagy or Type II Programmed Cell Death (PCD) is a major intracellular pathway for the degradation and recycling of proteins, ribosomes and entire organelles. The role of this pathway in the anti-tumor effect of radiotherapy and in radiation toxicity is obscure. A complicated machinery of genes and proteins is involved in the regulation of autophagy as a response to a variety of stress factors including hypoxia, nutrient deprivation, cytotoxic agents and radiotherapy. Continuously accumulating data suggest that autophagic response of cancer cells to radiotherapy is a major pathway which, in contrast to apoptosis that leads to death, may lead to either death or cellular survival. A variety of agents have been recognized that induce or block autophagy, directly interfering with the cytotoxic effect of radiotherapy. Simultaneous targeting of autophagy and apoptosis during radiotherapy seems to further augment the anti-tumor effect. Radiobiology research should focus on the differential effect of fractionation on the induction of autophagy in different tumors and on the manipulation of this with autophagy triggering agents. Whether manipulation of this pathway in normal tissues may be used to confer cytoportection deserves also thorough investigation. Moreover, the role of pretreatment autophagic indices in tumor cells in predicting radiotherapy and chemotherapy outcome should be examined in translational studies.     

Autophagic proteins: New facets of the oxygen paradox

Oxygen (O 2), while essential for aerobic life, can also cause metabolic toxicity through the excess generation of reactive oxygen species (ROS). Pathological changes in ROS production can originate through the partial reduction of O 2 during mitochondrial electron transport, as well as from enzymatic sources. This phenomenon, termed the oxygen paradox, has been implicated in aging and disease, and is especially evident in critical care medicine. Whereas high O 2 concentrations are utilized as a life-sustaining therapeutic for respiratory insufficiency, they in turn can cause acute lung injury. Alveolar epithelial cells represent a primary target of hyperoxia-induced lung injury. Recent studies have indicated that epithelial cells exposed to high O 2 concentrations die by apoptosis, or necrosis, and can also exhibit mixed-phenotypes of cell death (aponecrosis). Autophagy, a cellular homeostatic process responsible for the lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues. This evolutionarily conserved process is finely regulated by a complex interplay of protein factors. During autophagy, senescent organelles and cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded by lysosomal hydrolases, and the breakdown products released for reutilization in anabolic pathways. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states, and more generally, as a determinant of cell fate. However, the mechanisms by which autophagy and/or autophagic proteins potentially interact with and/or regulate cell death pathways during high oxygen stress, remain only partially understood.     

浮游植物细胞自噬作用特点及研究方法

自噬(Autophagy)是真核生物细胞中一类高度保守的、依赖于溶酶体或液泡途径对胞质蛋白和细胞器进行降解的生物学过程。细胞自噬除维持细胞稳态外,在细胞响应各种外界胁迫中也发挥重要作用。近年来,陆续发现浮游植物能够通过细胞自噬应答众多环境胁迫,并在浮游植物细胞中鉴定出了类似于哺乳动物细胞中的核心自噬功能单位。自噬作为一种独特的程序性细胞死亡(PCD)形式,对浮游植物遭受胁迫后的个体存活及种群延续具有至关重要的作用。因此,细胞自噬也将成为浮游植物研究领域的一个新的着力点。主要综述了浮游植物细胞中自噬的保守性、诱导因素、调控机制、自噬与凋亡的交互作用以及浮游植物自噬研究方法等研究进展。     

Targeting autophagy to fight hematopoietic malignancies

Macroautophagy, referred hereafter to as autophagy is an evolutionary conserved catabolic process for the degradation and recycling of macromolecules, bulk cytoplasm and dammaged organelles. Autophagy is activated under stress conditions induced by nutrient deprivation, hypoxia and drug treatments. Morphologically, autophagic cells are characterized by the accumulation of double membrane cytoplasmic vesicules called autophagosomes that surrounds cytoplasmic proteins and/or organelles. Autophagosomes next fuse with lysosomes to generate autolysosomes, the structures in which the retained constituents are digested before recycling into the cytoplasm. In this context, autophagy promotes cell survival under adverse conditions. In contrast, under certain circumstances autophagic cells may engage a specific mode of cell death called type II cell death or autophagic cell death (ACD). Considering the strategic positionnement of this process at the crossroads of cell death and survival, it is not surprising that defects in autophagy have been linked to a plethora of human diseases, including hematopoietic malignancies. Finally, autophagy induction is repressed by the mammalian target of rapamycin complex 1 (mTORC1) and favored by the adenosine-monophosphate activated-protein kinase (AMPK). In the present review, we focus on the functions of autophagy in normal and malignant hematopoiesis and discuss the opportunity to target the AMPK/mTOR pathways as a new therapeutic strategy to fight hematopoietic malignancies with a special emphasis on Chronic Myelogenous Leukemia (CML).     

Methods for monitoring autophagy

Autophagy is an intracellular bulk degradation system that is found ubiquitously in eukaryotes. Autophagy is responsible for the degradation of most long-lived proteins and some organelles. Cytoplasmic constituents, including organelles, are sequestered into double-membraned autophagosomes, which subsequently fuse with lysosomes where their contents are degraded. This system has been implicated in various physiological processes including protein and organelle turnover, the starvation response, cellular differentiation, cell death, and pathogenesis. However, methods for monitoring autophagy have been very limited and unsatisfactory. The most standard method is conventional electron microscopy. In addition, some biochemical methods have been utilized to measure autophagic activity. Recently, the molecular basis of autophagosome formation has been extensively studied using yeast cells; these studies have provided useful marker proteins for autophagosomes. Importantly, most of these proteins are conserved in mammals. Using these probes, we can now specifically monitor autophagic activity.     

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage.

Among the cellular responses to photodamage initiated by photodynamic therapy (PDT) are autophagy and apoptosis. While autophagy is a reversible process that can be both a survival and a death pathway, apoptosis is irreversible, leading only to cell death. In this study, we followed the fate of mouse leukemia L1210 cells after photodamage to the endoplasmic reticulum (ER) using a porphycene photosensitizer, where Bcl-2 was among the PDT targets. In wild-type cells, we observed a rapid wave of autophagy, presumed to represent the recycling of some damaged organelles, followed by apoptosis. Using shRNA technology, we created a Bax knockdown line (L1210/Bax(-)). In the latter cell line, we found a marked decrease in apoptosis after photodamage or pharmacologic inactivation of Bcl-2 function, but this did not affect PDT efficacy. Loss of viability was associated with a highly-vacuolated morphology consistent with autophagic cell death. Previous studies indicated pro-survival attributes of autophagy after low-dose PDT, suggesting that autophagy may be responsible for the 'shoulder' on the dose-response curve. It appears that attempts at extensive recycling of damaged organelles are associated with cell death, and that this phenomenon is amplified when apoptosis is suppressed.     

Apoptosis and Autophagy in Photoreceptors Exposed to Oxidative Stress

Studies on human and animal models of retinal dystrophy have suggested that apoptosis may be the common pathway of photoreceptor cell death. Autophagy, the major cellular degradation process in animal cells, is important in normal development and tissue remodeling, as well as under pathological conditions. Previously we provided evidence that genes, whose products are involved in apoptosis and autophagy, may be co-expressed in photoreceptors undergoing degeneration. Here, we investigated autophagy in oxidative stress-mediated cell death in photoreceptors, analyzing the light-damage mouse model and 661W photoreceptor cells challenged with H₂O₂. In the in vivo model, we demonstrated a time-dependent increase in the number of TUNEL-positive cells, concomitant with the formation of autophagosomes. In vitro, oxidative stress increased mRNA levels of apoptotic and autophagic marker genes. H₂O₂ treatment resulted in the accumulation of TUNEL-positive cells, the majority of which contain autophagosomes. To determine whether autophagy and apoptosis might precede each other or co-occur, we performed inhibitor studies. The autophagy inhibitor 3-methyladenine (3-MA), silencing RNA (siRNA) against two genes whose products are required for autophagy (autophagy-related (ATG) gene 5 and beclin 1), as well as the pan-caspase-3 inhibitor, zVAD-fmk, were both found to partially block cell death. Blocking autophagy also significantly decreased caspase-3 activity, whereas blocking apoptosis increased the formation of autophagosomes. The survival effects of 3-MA and zVAD-fmk were not additive; rather treatment with both inhibitors lead to increased cell death by necrosis. In summary, the study first suggests that autophagy participates in photoreceptor cell death possibly by initiating apoptosis. Second, it confirms that cells that normally die by apoptosis will execute cell death by necrosis if the normal pathway is blocked. And third, these results argue that the up-stream regulators of autophagy need to be identified as potential therapeutic targets in photoreceptor degeneration.