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.     

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.     

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.     

Chromaffin cell death induced by 6-hydroxydopamine is independent of mitochondrial swelling and caspase activation

Our results provide evidence that 6-hydroxydopamine induced, after auto-oxidation, toxic levels of hydrogen peroxide (H2O2) that caused bovine chromaffin cell toxicity and death. 6-Hydroxydopamine (6-OHDA) treatment markedly reduced, in a dose-response fashion, chromaffin cell viability. Cell death was accompanied by cell shrinkage, nuclear condensation and DNA degradation. Under our experimental conditions, 6-OHDA auto-oxidation formed quinones and reactive oxygen species (ROS) that mainly contributed to 6-OHDA-induced cytotoxicity in bovine chromaffin cells. Accordingly, different antioxidants, including catalase, vitamin E, Mn(IIItetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) or ascorbic acid, provided protection against 6-OHDA-induced toxicity. Further evidence that 6-OHDA induces oxidative stress is provided by the fact that this compound decreased total mitochondrial reduced NAD(P)H levels. Our results also suggest that mitochondrial swelling and caspase activation do not play a direct role in 6-OHDA-induced death in bovine chromaffin cells.     

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.     

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.     

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.     

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.     

Protein kinase C Inhibitors selectively modulate dynamics of cell adhesion molecules and cell death in human colon cancer cells

During development of colon cancer, Protein Kinase Cs (PKCs) are involved in regulation of many genes controlling several cellular mechanisms. Here, we examined the changes in cell adhesion molecules and PKCs for colorectal cancer progression. We identified that PKCs affected expression of EpCAM, claudins, tetraspanins. Treatment with low concentrations of PKC inhibitors resulted in decreased cell viability. In addition, immunoblotting and qRT-PCR analysis showed that apoptosis was inhibited while autophagy was induced by PKC inhibition in colon cancer cells. Furthermore, we observed decreased levels of intracellular Reactive Oxygen Species (ROS), lipid peroxidation and protein carbonyl, confirming the ROS-induced apoptosis. Taken together, our results reveal that PKC signalling modulates not only cell adhesion dynamics but also cell death-related mechanisms.

Abbreviations: PKC: Protein Kinase C; EpCAM: Epithelial cell adhesion molecule; FBS: fetal bovine serum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); CAM: cell adhesion molecule; ROS: reactive oxygen species     

Activation of DRD5 (dopamine receptor D5) inhibits tumor growth by autophagic cell death

Dopamine agonists such as bromocriptine and cabergoline have been successfully used in the treatment of pituitary prolactinomas and other neuroendocrine tumors. However, their therapeutic mechanisms are not fully understood. In this study we demonstrated that DRD5 (dopamine receptor D5) agonists were potent inhibitors of pituitary tumor growth. We further found that DRD5 activation increased production of reactive oxygen species (ROS), inhibited the MTOR pathway, induced macroautophagy/autophagy, and led to autophagic cell death (ACD) in vitro and in vivo. In addition, DRD5 protein was highly expressed in the majority of human pituitary adenomas, and treatment of different human pituitary tumor cell cultures with the DRD5 agonist SKF83959 resulted in growth suppression, and the efficacy was correlated with the expression levels of DRD5 in the tumors. Furthermore, we found that DRD5 was expressed in other human cancer cells such as glioblastomas, colon cancer, and gastric cancer. DRD5 activation in these cell lines suppressed their growth, inhibited MTOR activity, and induced autophagy. Finally, in vivo SKF83959 also inhibited human gastric cancer cell growth in nude mice. Our studies revealed novel mechanisms for the tumor suppressive effects of DRD5 agonists, and suggested a potential use of DRD5 agonists as a novel therapeutic approach in the treatment of different human tumors and cancers.     

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.     

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.     

Disruption of EARLY LESION LEAF 1, encoding a cytochrome P450 monooxygenase,induces ROS accumulation and cell death in rice

Lesion-mimic mutants (LMMs) provide a valuable tool to reveal the molecular mechanisms determining programmed cell death (PCD) in plants. Despite intensive research, the mechanisms behind PCD and the formation of lesions in various LMMs still remain to be elucidated. Here, we identified a rice (Oryza sativa) LMM, early lesion leaf 1 (ell1), cloned the causal gene by map-based cloning, and verified this by complementation. ELL1 encodes a cytochrome P450 monooxygenase, and the ELL1 protein was located in the endoplasmic reticulum. The ell1 mutant exhibited decreased chlorophyll contents, serious chloroplast degradation, upregulated expression of chloroplast degradation-related genes, and attenuated photosynthetic protein activity, indicating that ELL1 is involved in chloroplast development. RNA sequencing analysis showed that genes related to oxygen binding were differentially expressed in ell1 and wild-type plants; histochemistry and paraffin sectioning results indicated that hydrogen peroxide (H₂O₂) and callose accumulated in the ell1 leaves, and the cell structure around the lesions was severely damaged, which indicated that reactive oxygen species (ROS) accumulated and cell death occurred in the mutant. TUNEL staining and comet experiments revealed that severe DNA degradation and abnormal PCD occurred in the ell1 mutants, which implied that excessive ROS accumulation may induce DNA damage and ROS-mediated cell death in the mutant. Additionally, lesion initiation in the ell1 mutant was light dependent and temperature sensitive. Our findings revealed that ELL1 affects chloroplast development or function, and that loss of ELL1 function induces ROS accumulation and lesion formation in rice.     

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.     

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.     

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l -cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factor кB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.     

FER1L4/miR‐372/E2F1 works as a ceRNA system to regulate the proliferation and cell cycle of glioma cells

Long non‐coding RNAs have recently become a key regulatory factor for cancers, whereas FER1L4, a newly discovered long non‐coding RNA, has been mostly studied in gastric carcinoma and colon cancer cases. The functions and molecular mechanism of FER1L4 have been rarely reported in glioma malignant phenotypes. In this study, it was found that the expression of LncRNA FER1L4 is upregulated in high‐grade gliomas than in low‐grade cases and that a high expression of LncRNA FER1L4 predicts poor prognosis of gliomas. Meanwhile, in vitro study suggests that expression of FER1L4 with SiRNA knockdown obviously suppresses cell cycle and proliferation. It is further demonstrated by experiments that the FER1L4 knockdown suppresses growth of in vivo glioma. Besides, it is found in our study that LncRNA FER1L4 expression is positively correlated with E2F1 mRNA expression. After knockdown of FER1L4 expression, E2F1 expression is significantly down‐regulated, whereas the expression of miR‐372 is significantly up‐regulated; the up‐regulation of miR‐372 leads to significant down‐regulation of FER1L4 and E2F1 expression. In addition, it is also found that FER1L4 can be used as competitive endogenous RNA to interact or bind with miR‐371 and thereby up‐regulate E2F1, thus promoting the cycle and proliferation of glioma cells. It may be one of the molecular mechanisms in which FER1L4 plays its oncogene‐like role in gliomas.