E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells

The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.     

Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress

Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species(ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membranebound vacuoles characteristic of autophagy followed by autophagic cell death(referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.     

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.     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

High dose of ascorbic acid induces cell death in mesothelioma cells

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.     

Cross-talk between pRb/E2F and Wnt/beta-catenin pathways: E2F1 induces axin2 leading to repression of Wnt signalling and to increased cell death

The pRb/E2F and Wnt/beta-catenin pathways are two of the most frequently deregulated pathways in human cancers. In this study, we show that E2F1 up-regulates the expression of axin2. Further, we show that axin2 can repress Wnt signalling leading to reduced cell growth and increased cell death. This represents cross-talk between major pathways involved in the formation of tumours. We use our data to suggest a novel mechanism for tumour suppression.     

Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.     

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin-regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1-induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-gamma. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.     

Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant programmed cell death induced by ultraviolet-C overexposure

Recent studies have suggested that ultraviolet-C (UV-C) overexposure induces programmed cell death (PCD) in Arabidopsis thaliana (L.) Heynh, and this process includes participation of caspase-like proteases, DNA laddering as well as fragmentation of the nucleus. To investigate possible early signal events, we used microscopic observations to monitor in vivo the behaviour of mitochondria, as well as the production and localization of reactive oxygen species (ROS) during protoplast PCD induced by UV-C. A quick burst of ROS was detected when the protoplasts were kept in continuous light after UV-C exposure, which was restricted in chloroplasts and the adjacent mitochondria. Pre-incubation with ascorbic acid (AsA, antioxidant molecule) or 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU, an inhibitor of photosynthetic electron transport) decreased the ROS production and partially protected protoplasts from PCD. A mitochondrial transmembrane potential (MTP) loss occurred prior to cell death; thereafter, the mitochondria irregularly clumped around chloroplasts or aggregated in other places within the cytoplasm, and the movement of mitochondria was concomitantly blocked. Pre-treatment with an inhibitor of mitochondrial permeability transition pores (MPTP), cyclosporine (CsA), effectively retarded the decrease of MTP and reduced the percentage of protoplasts undergoing PCD after UV-C overexposure. Our results suggest that the MTP loss and the changes in distribution and mobility of mitochondria, as well as the production of ROS play important roles during UV-induced plant PCD, which is in good accordance with what has been reported in many types of apoptotic cell death, both in animals and plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteomic analysis of the cellular responses induced in uninfected immune cells by cell-expressed X4 HIV-1 envelope

HIV-1 envelope gp120 and gp41 glycoproteins (Env), expressed at the cell surface, induce uninfected CD4 T-cell death, but the molecular mechanisms leading to this demise are still largely unknown. To better understand these events, we analyzed by a proteomic approach the differential protein expression profile of two types of uninfected immune cells after their coculture for 1-3 days with cells that express, or not, Env. First, umbilical cord blood mononuclear cells (UCBMCs) were used to approach the in vivo situation, i.e., blood uninfected naive cells that encounter infected cells. Second, we used the A2.01/CD4.403 T-cell line expressing wild type CXCR4 and a truncated form of CD4 that still undergoes Env-mediated apoptosis, independently of CD4 signaling. After coculture with cells expressing Env, 35 and 39 proteins presenting an altered expression in UCBMCs and the A2.01/CD4.403 T-cell line, respectively, were identified by mass-spectrometry. Whatever the cell type analyzed, the majority of these proteins are involved in degradation processes, redox homeostasis, metabolism and cytoskeleton dynamics, and linked to mitochondrial functions. This study provides new insights into the events that sequentially occur in bystander T lymphocytes after contact with HIV-infected cells and leading, finally, to apoptotic cell death.     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.     

The atypical pattern of cell death in B16F10 melanoma cells treated with TNP-470

TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.     

4-Hydroxynonenal affects pRb/E2F pathway in HL-60 human leukemic cells

4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation, has an antiproliferative effect in several tumor cell lines and provokes alteration of cell cycle progression in HL-60 cells. HNE down-regulates c-myc expression in K562, HL-60, and MEL cells. This prompted us to study the cascade of phenomena that, starting from the CKIs expression and the phosphorylation of pRb, arrives at the E2F binding to consensus sequence in the P2 promoter of the c-myc gene. Treatment of HL-60 cells with HNE (1 microM) causes a p53-independent increase of p21(WAF1/CIP1) expression, pRb dephosphorylation, a decrease of low molecular weight E2F complexes and an increase of high molecular weight E2F complexes bound to P2 c-myc promoter. E2F4 expression is reduced by HNE treatment as well as the amount of pRb/E2F4 complexes, whereas the amount of pRb/E2F1 complexes is increased. In conclusion, HNE can affect the pRb/E2F pathway by modifying the expression of several genes involved in the control of cell proliferation.     

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A₁, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.     

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death,is necessary for developmental cell death in Drosophila midgut

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported¹ Dasari SK, Bialik S, Levin-Zaidman S, Levin-Salomon V, Merrill AH, Jr., Futerman AH, Kimchi A. Signalome-wide rnai screen identifies gba1 as a positive mediator of autophagic cell death. Cell Death Differ. 2017;24(7):1288-1302. https://doi.org/10.1038/cdd.2017.80. PMID:28574511[Crossref], [PubMed], [Web of Science ®] [Google Scholar] on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.     

Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species

The fungal AAL-toxin triggers programmed cell death (PCD) through perturbations of sphingolipid metabolism in AAL-toxin-sensitive plants. While Arabidopsis is relatively insensitive to the toxin, the loh2 mutant exhibits increased susceptibility to AAL-toxin due to the knockout of a gene involved in sphingolipid metabolism. Genetic screening of mutagenized loh2 seeds resulted in the isolation of AAL-toxin-resistant mutant atr1.Atr1 displays a wild type phenotype when grown on soil but it develops less biomass than loh2 on media supplemented with 2% and 3% sucrose. Atr1 was also more tolerant to the reactive oxygen species-generating herbicides aminotriazole (AT) and paraquat. Microarray analyses of atr1 and loh2 under AT-treatment conditions that trigger cell death in loh2 and no visible damage in atr1 revealed genes specifically regulated in atr1 or loh2. In addition, most of the genes strongly downregulated in both mutants were related to cell wall extension and cell growth, consistent with the apparent and similar AT-induced cessation of growth in both mutants. This indicates that two different pathways, a first controlling growth inhibition and a second triggering cell death, are associated with AT-induced oxidative stress.     

Nucleus image properties and cell death in MCF-10F cells grown on slide substrates differing in nature and size

Summary The immortalized human breast epithelial cell line MCF-10F is an important tool for studies on experimental tumorigenesis induced by drugs, transfected Ha-ras oncogene, and hormones. Considering that many relevant data have thus far been established only for MCF-10F cells cultivated on glass, and that there are data showing different cell death ratios for tumorigenic cells obtained from benzo[a]pyrene (BP)-transformed MCF-10F cells cultivated on plastic compared with glass, nuclear parameters estimated by image analysis and cell death ratios were compared for cells grown on plastic and glass substrates differing in chamber surface sizes and working culture medium volumes. It was concluded that for slides with a growth size equal to 9.4 cm², plastic substrate was more advantageous than glass for growing MCF-10F cells because although the apoptotic ratios (AR) for the cells grown on plastic are low as it would be expected for nontransformed cells, they are bigger than those reported for the BP-transformed MCF-10F cells cultivated on the same substrate but closer to those of the BP-transformed MCF-10F cells receiving a normal chromosome 17. In addition, the plastic substrate did not induce variable nuclear image results as those found in the latter. The 0.5-cm²-sized chambers on plastic slides proved to be inadequate for cell nuclear image analysis and cell death studies on account of the variable geometric, densitometric, and textural results and ARs produced and the unpublished consideration of a very slow growth rate generated under this growth condition.