Stages Of Autophagy Research Articles

Chloroquine affects autophagy to achieve an anticancer effect in EC109 esophageal carcinoma cells in vitro.

Esophageal carcinoma is a malignancy that severely threatens human health, with a high incidence rate and a low 5-year survival rate. Resistance to chemotherapy frequently emerges during its treatment, partly due to the induction of autophagy. Therefore, targeting autophagy may be a promising therapeutic approach for the treatment of esophageal carcinoma. In the present study, it was investigated how chloroquine (CQ) can influence the growth ability and biological behaviors of EC109 esophageal squamous carcinoma cells in vitro, as well as the potential molecular mechanisms behind its activity. It was demonstrated that CQ could suppress the growth and proliferation of EC109 cells in a time- and dose-dependent manner; migration and colony formation abilities were also inhibited by CQ. Furthermore, subsequent to the exposure to CQ, the number of autophagosomes was clearly increased in EC109 cells overexpressing green fluorescent protein tagged-light chain (LC)3 when observed by fluorescence microscopy. Protein expression of the endogenous autophagy markers LC3-II and p62 was elevated subsequent to CQ treatment, whereas the expression of proteins from the protein kinase B/mechanistic target of rapamycin target of rapamycin pathway was inhibited. This suggested that CQ could induce the formation of autophagosomes in the initiation of autophagy, but inhibit the degradation of autophagosomes in a later stage of autophagy. The overall effect was that autophagic cell death was activated by CQ, as confirmed by flow cytometry. Overall, the anticancer effect of chloroquine on EC109 was revealed to be mediated through modulating autophagy, and this may produce promising therapeutic benefits for esophageal carcinoma.

Impaired placental autophagy in placental malaria.

BackgroundPlacental malaria is a major cause of low birthweight, principally due to impaired fetal growth. Intervillositis, a local inflammatory response to placental malaria, is central to the pathogenesis of poor fetal growth as it impairs transplacental amino acid transport. Given the link between inflammation and autophagy, we investigated whether placental malaria-associated intervillositis increased placental autophagy as a potential mechanism in impaired fetal growth.MethodsWe examined placental biopsies collected after delivery from uninfected women (n = 17) and from women with Plasmodium falciparum infection with (n = 14) and without (n = 7) intervillositis. Western blotting and immunofluorescence staining coupled with advanced image analysis were used to quantify the expression of autophagic markers (LC3-II, LC3-I, Rab7, ATG4B and p62) and the density of autophagosomes (LC3-positive puncta) and lysosomes (LAMP1-positive puncta).ResultsPlacental malaria with intervillositis was associated with higher LC3-II:LC3-I ratio, suggesting increased autophagosome formation. We found higher density of autophagosomes and lysosomes in the syncytiotrophoblast of malaria-infected placentas with intervillositis. However, there appear to be no biologically relevant increase in LC3B/LAMP1 colocalization and expression of Rab7, a molecule involved in autophagosome/lysosome fusion, was lower in placental malaria with intervillositis, indicating a block in the later stage of autophagy. ATG4B and p62 expression showed no significant difference across histological groups suggesting normal autophagosome maturation and loading of cargo proteins into autophagosomes. The density of autophagosomes and lysosomes in the syncytiotrophoblast was negatively correlated with placental amino acid uptake.ConclusionsPlacental malaria-associated intervillositis is associated with dysregulated autophagy that may impair transplacental amino acid transport, possibly contributing to poor fetal growth.

Vitamin D/vitamin D receptor, autophagy and inflammation relevant diseases

The biological effects mediated by vitamin D and vitamin D receptor (VDR) are involved in the regulation of multiple pathophysiologic processes, including calcium phosphorus metabolism, immune regulation, anti-inflammation, anti-infection and cancer prevention, etc. Autophagy is a eukaryotic cell lysosome-mediated catabolic pathway, which is classified into 3 different types: macroautophagy, microautophagy, and chaperone-mediated autophagy. Since autophagy is vital to maintain energy and metabolism in cells, autophagy dysfunction is closely associated with various pathological processes such as inflammation, infection and tumor. Studies have shown that vitamin D/VDR can affect the different stages of autophagy and regulate inflammation in autophagy. Moreover, vitamin D/VDR and autophagy play an important role in autoimmune disease, diabetes, cardiovascular disease, cancer and other inflammatory diseases.

Autophagy: an adaptive physiological countermeasure to cellular senescence and ischaemia/reperfusion-associated cardiac arrhythmias.

Oxidative stress placed on tissues that involved in pathogenesis of a disease activates compensatory metabolic changes, such as DNA damage repair that in turn causes intracellular accumulation of detritus and ‘proteotoxic stress’, leading to emergence of ‘senescent’ cellular phenotypes, which express high levels of inflammatory mediators, resulting in degradation of tissue function. Proteotoxic stress resulting from hyperactive inflammation following reperfusion of ischaemic tissue causes accumulation of proteinaceous debris in cells of the heart in ways that cause potentially fatal arrhythmias, in particular ventricular fibrillation (VF). An adaptive response to VF is occurrence of autophagy, an intracellular bulk degradation of damaged macromolecules and organelles that may restore cellular and tissue homoeostasis, improving chances for recovery. Nevertheless, depending on the type and intensity of stressors and inflammatory responses, autophagy may become pathological, resulting in excessive cell death. The present review examines the multilayered defences that cells have evolved to reduce proteotoxic stress by degradation of potentially toxic material beginning with endoplasmic reticulum‐associated degradation, and the unfolded protein response, which are mechanisms for removal from the endoplasmic reticulum of misfolded proteins, and then progressing through the stages of autophagy, including descriptions of autophagosomes and related vesicular structures which process material for degradation and autophagy‐associated proteins including Beclin‐1 and regulatory complexes. The physiological roles of each mode of proteotoxic defence will be examined along with consideration of how emerging understanding of autophagy, along with a newly discovered regulatory cell type called telocytes, may be used to augment existing strategies for the prevention and management of cardiovascular disease.

The Bioflavinoid Quercetin Induces Apoptosis and Inhibits Autophagy in Primary Acute Lymphoblastic Leukemia in-Vitro

Quercetin is an antioxidant previously shown to inhibit acute lymphoblastic leukemia (ALL) growth and induce both apoptosis and autophagy in malignant hematologic cell lines derived from AML and T-ALL. Since autophagy is a critical mechanism in the response to cellular stress, we hypothesized that high-risk ALL cells had higher resting autophagy than standard risk ALL cells. To test our hypothesis, we quercetin-treated stable cell cultures of ALL isolated from the diagnostic marrow specimens of one clinically high-risk (HR-ALL, an infant carrying the t(4;11) MLL-rearrangement) and one cytogenetically normal standard-risk pediatric patient (SR-ALL), and compared viability and the expression of molecular markers of programmed cell death and autophagy by Western Blot.First, we established a dose-response curve that identified 50 uM quercetin as the target dose for potency and efficacy in HR- and SR-ALL. Next, we compared viability between HR-and SR-ALL treated with 50 uM quercetin at 3, 12, 24 and 48 hours. HR-ALL treated with quercetin exhibited a significantly greater reduction in viability measured by Annexin V/PI staining and MTT reagent conversion (2-way ANOVA p = 0.04 and p = 0.005). HR-ALL cells also exhibited greater catalytic cleavage and activation programmed death markers, Caspase 3 and PARP1 by Western Blot, compared to SR-ALL.Because high levels of autophagy can induce cell death, we asked whether HR-ALL cells had a higher resting autophagy rate. We compared expression of the prototypic autophagy protein, Beclin-1 at 30min, 1hr, 2hr, 3hr, and 4hr after treatment with 10mM of the autophagy inhibitor 3-methyladenine (3-MA). Because 3-MA inhibits autophagy at the early stage of nucleation, the rate of breakdown of downstream proteins such as Beclin-1 is a proxy measure for the rate of resting, non-stress induced autophagy. HR-ALL cells exhibited a significantly greater rate of Beclin-1 breakdown after 3-MA treatment as compared to SR-ALL cells (slope -0.235 vs. 0.131, p = 0.003) consistent with a higher resting autophagy rate in HR-ALL.To determine whether quercetin treatment impacts this higher rate of autophagy, we treated HR-ALL and SR-ALL cells with 50uM of quercetin for 24hrs and compared, by Western blot, the expression of regulatory proteins at each stage of autophagy: nucleation (ATG7, Beclin-1), expansion (ATG5, ATG16L1) and maturation (LC3A/B). Unexpectedly, quercetin inhibited expression of all autophagy proteins, with a greater decrease in HR-ALL. We next parsed whether selective autophagy inhibition at formation of the autophagosome, using 3MA, or later endosome acidification, using chloroquine, could explain the differential cytotoxicity of quercetin in HR-ALL. 3MA and chloroquine treatment alone resulted in significantly fewer Annexin+/PI+ HR- and SR-ALL cells and less MTT conversation compared to quercetin treatment alone (p<0.05) suggesting that selective inhibition of the autophagy complex was insufficient to explain quercetin cytotoxicity.Finally, we asked whether differences in an upstream regulator of the autophagy/apoptotic balance contribute to higher quercetin toxicity in HR-ALL cells. The regulatory protein high mobility group box-1 (HMGB1) has been shown to induce autophagy by interacting with Beclin-1, and we have previously demonstrated overexpression of HMGB1 in HR-ALL. We verified interaction of HMGB1 with Beclin-1 by immunoprecipitation, and observed a substantial reduction in complex formation after 50 uM quercetin treatment in HR-ALL cells. (1511153 vs. 70442 Beclin-1 band volumes after IP p<0.001), along with decreased expression of the anti-apoptotic proteins, Bcl-2 and Mcl-1, factors proposed to be regulated by the Beclin-1/HMGB1 complex.Taken together, these data indicate that regulation of the autophagy-apoptotic balance is a mechanism used by ALL cells to mitigate cellular stress, and is the first to describe the effects of quercetin in the context of autophagy in ALL. Future studies will compare the expression profile of critical autophagy proteins following chemotherapy treatment in a larger cohort of high-risk ALL cells, and delineate the role of HMGB1 as an autophagy regulator using our established knockdown model. DisclosuresNo relevant conflicts of interest to declare.

Abnormal cell-clearance and accumulation of autophagic vesicles in lymphocytes from patients affected with Ataxia-Teleangiectasia

Ataxia-Teleangiectasia (A-T) is a neurodegenerative disorder due to mutations in ATM gene. ATM in the nucleus ensures DNA repair, while its role in the cytosol is still poorly clarified. Abnormal autophagy has been documented in other neurodegenerative disorders, thus we evaluated whether alteration in this process may be involved in the pathogenesis of A-T by analyzing the autophagic vesicles and the genes implicated in the different stages of autophagy. Through transmission electron microscopy (TEM) and immunofluorescence analysis we observed an accumulation of APs associated with a LC3 puncta pattern, and a reduced number of ALs. We also documented an increased expression of genes involved in AP and lysosome biogenesis and function, and a decrease of Vps18 expression, involved in their vesicular trafficking and fusion. mTORC1-controlled proteins were hyperphosphorylated in A-T, in keeping with an increased mTOR inhibitory influence of autophagy. Betamethasone is able to promote the degradation of SQSTM1, a biomarker of autophagy. Collectively, our results indicate that in cells from A-T patients, the APs maturation is active, while the fusion between APs and lysosomes is inappropriate, thus implying abnormalities in the cell-clearance process. We also documented a positive effect of Betamethasone on molecules implicated in autophagosome degradation.

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

ABSTRACTThe N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.

A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy.

The intronic GGGGCC hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is a prevalent genetic abnormality identified in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) is a protein with unclear functions. We report that C9ORF72 is a component of a multiprotein complex containing SMCR8, WDR41, and ATG101 (an important regulator of autophagy). The C9ORF72 complex displays guanosine triphosphatase (GTPase) activity and acts as a guanosine diphosphate-guanosine 5'-triphosphate (GDP-GTP) exchange factor (GEF) for RAB39B. We created Smcr8 knockout mice and found that Smcr8 mutant cells exhibit impaired autophagy induction, which is similarly observed in C9orf72 knockdown cells. Mechanistically, SMCR8/C9ORF72 interacts with the key autophagy initiation ULK1 complex and regulates expression and activity of ULK1. The complex has an additional role in regulating later stages of autophagy. Whereas autophagic flux is enhanced in C9orf72 knockdown cells, depletion of Smcr8 results in a reduced flux with an abnormal expression of lysosomal enzymes. Thus, C9ORF72 and SMCR8 have similar functions in modulating autophagy induction by regulating ULK1 and play distinct roles in regulating autophagic flux.

Regulation of actin nucleation and autophagosome formation.

Autophagy is a process of self-eating, whereby cytosolic constituents are enclosed by a double-membrane vesicle before delivery to the lysosome for degradation. This is an important process which allows for recycling of nutrients and cellular components and thus plays a critical role in normal cellular homeostasis as well as cell survival during stresses such as starvation or hypoxia. A large number of proteins regulate various stages of autophagy in a complex and still incompletely understood series of events. In this review, we will discuss recent studies which provide a growing body of evidence that actin dynamics and proteins that influence actin nucleation play an important role in the regulation of autophagosome formation and maturation.

High glucose induces autophagy of MC3T3-E1 cells via ROS-AKT-mTOR axis

In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis, proliferation and autophagy of MC3T3-E1 cells through ROS-AKT-mTOR axis. In addition, autophagy inhibition does not affect the proliferation of MC3T3-E1 cells under high glucose conditions.

Autophagic flux in glioblastoma cells

To establish metabolic context for radiation sensitivity by measuring autophagic flux in two different glioblastoma (GBM) cell lines. Clonogenic survival curve analysis of U87 or U251 cells exposed to γ radiation, fast neutrons, a mixed energy neutron beam (METNB) or Auger electrons from a gadolinium neutron capture (GdNC) reaction suggested other factors, beyond a defective DNA damage response, contribute to cell death of U251 cells. Altered tumor metabolism (autophagy) was hypothesized as a factor in U251 cells’ clonogenic response. Each of the four different radiation modalities induced an increase in the number of autophagosomes in both U87 and U251 cells. Changes in the number of autophagosomes can be explained by either induction of autophagy or alterations in autophagic flux so autophagic flux was assayed by p62 immunoblotting or in engineered GBM cells that stably express an autophagy marker protein, LC3B-eGFP-mCherry. Perturbations in later stages of autophagy in U251 cells corresponded with radiation sensitivity of U251 cells irradiated with 10 Gy γ rays. Establishment of altered autophagic flux is a useful biomarker for metabolic stress and provided metabolic context for radiation sensitization to 10 Gy γ rays. These results provide strong evidence for the usefulness of managing tumor cell metabolism as a tool for the enhancement of radiation therapy.

Abstract 5412: Lysosome disruption rapidly and effectively induces necrosis in aggressive thyroid carcinomas

Abstract Poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are very aggressive and lethal neoplasms for which there is currently no effective therapy. By activating Kras and deleting p53 in the mouse thyroid we have generated a mouse model of PDTC progressing to ATC. These tumors are characterized by elevated levels of anti-apoptotic members of the Bcl2 family. Tumor cell lines established from this model as well as similarly aggressive human thyroid cancer cell lines undergo cell death upon treatment with Obatoclax, an inhibitor of the anti-apoptotic Bcl2 family members. Even though several studies have suggested that Obatoclax induces apoptosis or necroptosis by binding to its designated targets in a number of different cancer models, our studies show that Obatoclax induces necrosis in both human and mouse thyroid cancer cell lines. By exploiting its autofluorescence, we show that Obatoclax is found in association with lysosomes, but not with mitochondria as it would be expected given the location of its known Bcl2-family targets, and that it causes rapid lysosome disruption. Obatoclax was also able to induce a block in the later stages of autophagy, as shown by an accumulation of autophagosomes upon treatment, likely as a result of lysosome impairment. Furthermore, Bafilomycin A1, an inhibitor of lysosomal acidification, counteracts the effect of Obatoclax and allows for cell survival. These findings suggest a novel mechanism of action for Obatoclax, in which direct lysosome disruption leads to necrosis. Strikingly, human and mouse thyroid cancer cell lines are also sensitive to other lysosome-destabilizing agents. Importantly, transformed human thyroid cell lines are much more sensitive to Obatoclax than non-transformed thyroid cells, suggesting the existence of a useful therapeutic window. Even though Obatoclax has been removed from clinical trials due to low anti-tumor activity and toxic side effects, its efficacy in killing thyroid cancer cells otherwise refractory to treatment, and the alternative mechanism of action involved in this process support a new approach for targeting aggressive thyroid neoplasms. In conclusion, our data show that aggressive thyroid cancer cells are intrinsically sensitive to lysosome destabilization, and this characteristic can be exploited for the development of novel therapeutic approaches. Citation Format: Devora Champa, Antonio Di Cristofano. Lysosome disruption rapidly and effectively induces necrosis in aggressive thyroid carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5412. doi:10.1158/1538-7445.AM2015-5412

ATG16L1 expression in carotid atherosclerotic plaques is associated with plaque vulnerability

Objective— Autophagy has emerged as a cell survival mechanism critical for cellular homeostasis, which may play a protective role in atherosclerosis. ATG16L1, a protein essential for early stages of autophagy, has been implicated in the pathogenesis of Crohn’s disease. However, it is unknown whether ATG16L1 is involved in atherosclerosis. Our aim was to analyze ATG16L1 expression in carotid atherosclerotic plaques in relation to markers of plaque vulnerability. Approach and Results— Histological analysis of 143 endarterectomized human carotid atherosclerotic plaques revealed that ATG16L1 was expressed in areas surrounding the necrotic core and the shoulder regions. Double immunofluorescence labeling revealed that ATG16L1 was abundantly expressed in phagocytic cells (CD68), endothelial cells (CD31), and mast cells (tryptase) in human advanced plaques. ATG16L1 immunogold labeling was predominantly observed in endothelial cells and foamy smooth muscle cells of the plaques. ATG16L1 protein expression correlated with plaque content of proinflammatory cytokines and matrix metalloproteinases. Analysis of Atg16L1 at 2 distinct stages of the atherothrombotic process in a murine model of plaque vulnerability by incomplete ligation and cuff placement in carotid arteries of apolipoprotein-E-deficient mice revealed a strong colocalization of Atg16L1 and smooth muscle cells only in early atherosclerotic lesions. An increase in ATG16L1 expression and autophagy flux was observed during foam cell formation in human macrophages using oxidized-LDL. Conclusions— Taken together, this study shows that ATG16L1 protein expression is associated with foam cell formation and inflamed plaque phenotype and could contribute to the development of plaque vulnerability at earlier stages of the atherogenic process.

Abstract 567: Atg16l1 Expression in Carotid Atherosclerotic Plaques Is Associated With Plaque Vulnerability

Objective: Autophagy has emerged as a cell survival mechanism critical for cellular homeostasis, which may play a protective role in atherosclerosis. ATG16L1, a protein essential for early stages of autophagy, has been implicated in the pathogenesis of inflammatory diseases such as Crohn’s disease. However, it is unknown whether ATG16L1 is involved in atherosclerosis. Our aim was to analyze ATG16L1 expression in carotid atherosclerotic plaques in relation to markers of plaque vulnerability. Approach and results: Histological analysis of 143 endarterectomized human carotid atherosclerotic plaques revealed that ATG16L1 was expressed in areas surrounding the necrotic core and the shoulder regions. Double immunofluorescence labelling for ATG16L1 and cell type markers CD68, α-actin-smooth muscle cell, CD31 or tryptase revealed that ATG16L1 was abundantly expressed in phagocytic cells, endothelial cells, as well as mast cells in human advanced carotid plaques. ATG16L1 immunogold labelling was predominantly observed in endothelial cells and foamy smooth muscle cells of the plaques. ATG16L1 protein expression correlated with plaque content of pro-inflammatory cytokines and matrix metalloproteinases. Analysis of Atg16L1 at two distinct stages of the atherothrombotic process in a murine model of plaque rupture by incomplete ligation and cuff placement in carotid arteries of apolipoprotein-E-deficient mice, revealed a strong colocalization of Atg16L1 and smooth muscle cells only in early atherosclerotic lesions developing intimal hyperplasia. Conclusions: Taken together, this study shows that ATG16L1 protein expression is associated with a rupture-prone and inflamed plaque phenotype, and could also contribute to the development of plaque vulnerability already at earlier stages of the atherogenic process.

Promoting autophagic clearance: viable therapeutic targets in Alzheimer's disease.

Many neurodegenerative disorders are characterized by the aberrant accumulation of aggregate-prone proteins. Alzheimer's disease (AD) is associated with the buildup of β-amyloid peptides and tau, which aggregate into extracellular plaques and neurofibrillary tangles, respectively. Multiple studies have linked dysfunctional intracellular degradation mechanisms with AD pathogenesis. One such pathway is the autophagy-lysosomal system, which involves the delivery of large protein aggregates/inclusions and organelles to lysosomes through the formation, trafficking, and degradation of double-membrane structures known as autophagosomes. Converging data suggest that promoting autophagic degradation, either by inducing autophagosome formation or enhancing lysosomal digestion, provides viable therapeutic strategies. In this review, we discuss compounds that can augment autophagic clearance and may ameliorate disease-related pathology in cell and mouse models of AD. Canonical autophagy induction is associated with multiple signaling cascades; on the one hand, the best characterized is mammalian target of rapamycin (mTOR). Accordingly, multiple mTOR-dependent and mTOR-independent drugs that stimulate autophagy have been tested in preclinical models. On the other hand, there is a growing list of drugs that can enhance the later stages of autophagic flux by stabilizing microtubule-mediated trafficking, promoting lysosomal fusion, or bolstering lysosomal enzyme function. Although altering the different stages of autophagy provides many potential targets for AD therapeutic interventions, it is important to consider how autophagy drugs might also disturb the delicate balance between autophagosome formation and lysosomal degradation.

Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings.

AB77. Tetrandrine blocks autophagic flux by preventing lysosomal acidification in 786-O cells

Introduction and objectivesAutophagy is a highly regulated catabolic process that in cancer cells maintains metabolism and energy balances, both of which protect malignant cells from stress. Lysosomes are acidic organelles that play a crucial role in degrading intracellular macromolecules and organelles during the final stage of autophagy. Tetrandrine (Tet) is a natural product isolated from the root of Stephania tetrandra S. Moore. Recently, Tet was reported to trigger apoptosis and cell cycle arrest in human renal carcinoma cells. However, the detailed anti-tumor mechanism of Tet remains unclear. Here, we show that Tet is a potent and rapid lysosomal deacidification agent and single-agent Tet exhibits powerful antitumor activity in vitro.Methods786-O cells were used to evaluate the anti-tumor effects of Tet. The lipidation levels of microtubuleassociated protein 1 light chain 3 (LC3) and expression levels of sequestosome 1 (p62) were detected by Western blotting. Apoptosis, mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. LC3 turnover assays were performed to assess autophagy flux. Oxidative phosphorylation (OXPHOS) was measured using the Seahorse XF24 analyzer. Lysosomal pH was detected using LysoSensor DND-160.ResultsTet exhibits powerful antitumor activity in vitro. Tet induces significant apoptosis and decrease of ΔΨm in 786-O cells. In the presence of Tet, apoptosis was preceded by a robust accumulation of autophagosomes and increased level of LC3-II. However, Tet simultaneously increased the level of p62 and decreased LC3 turnover, indicating the blockade of autophagic flux in the degradation stage. Moreover, the blockade of autophagy is mainly caused by its effect on neutralizing lysosomal acidity. Tetmediated inhibition of autophagy leads to insufficient substrates for tricarboxylic acid (TCA) cycle, impaired oxidative phosphorylation, reduction of ATP and activation of AMPKα. Blunting autophagosome formation with 3-methyladenine or genetically knocking down Beclin-1 did not rescue cells upon Tet treatment. By contrast, the addition of methyl pyruvate to supplement TCA substrates partially protected Tet-treated 786-O cells. Taken together, our results suggest that Tet blocks autophagic flux by promoting lysosomal deacidification, leading to energy depletion and caspase-mediated apoptosis.ConclusionsOur research recommends the evaluation of Tet as a potential cancer chemotherapeutic drug, based on its demonstrated ability to inhibit autophagy.

Autophagic degradation of CD81 by hepatitis C virus: a novel mechanism underlying viral superinfection exclusion (740.1)

Autophagy, a lysosomal degradation pathway, is known to positively regulate HCV replication. However, it remains unknown how cells withstand HCV productive replication in the cell context of autophagy activation. Here we showed that HCV infection triggered superinfection exclusion through downregulating the cell surface and total expressions of CD81, an important HCV entry (co)receptor. HCV‐induced downregulation of CD81 was associated with the autophagy‐mediated, lysosomal degradation process as interference with autophagolysosome maturation with pharmacological inhibitors restored the level of CD81 in HCV‐infected cells. Moreover, confocal and electron microscopy analyses showed that HCV infection led to engulfment of CD81 within autophagic vacuoles matured at different stages of autophagy, and that these CD81‐coresiding autophagosomes were originated from endoplasmic reticulum (ER)‐derived preautophagosomal structure (PAS). Knockdown of autophagy‐related genes required for the assembly of PAS in HCV‐infected cells relieved cytoplasmic retention of CD81, restored CD81 surface expression, and re‐conferred cell permissiveness to HCV infection. Our findings reveal a novel role of autophagy in restricting HCV superinfection by sequestration and degradation of its entry (co)receptor through ER‐originated PAS and fusion with lysosome, thereby maintaining virus‐cell homeostasis.

Regulation of autophagy by the Rab GTPase network

Autophagy (macroautophagy) is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy. Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B participate in autophagosome formation, whereas Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are also involved in autophagy, but their role is unknown. Here, we summarize new findings regarding the involvement of Rabs in autophagy and provide insights regarding future research on the mechanisms of autophagy regulation.

Autophagy activity is inhibited in human fibroblast cells stably overexpressing H-RasV 12

Objective To investigate the relationship between oncogenic H-RasV12 overexpression/activation and the autophagic activity by observing the effect of Ras overexpression on autophagic activity in human fibroblast cells.MethodsHuman BJ fibroblast cells were transfected with H-RasV12 or control vector,and then the cellular responses to H-RasV12 overexpression were analyzed by observing the morphology,cell growth curve,senescence-associatedβ-Gal staining,Western blotting analysis,flow cytometry,and suppression of autophagy-related protein 5(ATG5)by siRNA.Results Compared with control group,BJ cells overexpressing H-RasV12 developed prominent premature senescence and inhibited autophagic activity,as manifested by significant accumulation of p62 and light chain 3Ⅱ(LC3Ⅱ).The autophagy inhibition by H-RasV12 remained stable during the study period;the apoptosis rate was increased in H-RasV12 overexpressing BJ cells compared with that in the control cells.Suppression of ATG5 by siRNA led to more severe senescence in Ras-overexpressing BJ cells.Conclusion Our results suggest that the autophagy activity is inhibited in human fibroblast cells stably overexpressing oncogenic H-RasV12,and the inhibition is in the later stage of autophagy,which may be related to H-RasV12-related tumorigenesis.