WorldWideScience

Sample records for autophagy genes protect

  1. Determination of autophagy gene ATG16L1 polymorphism in human colorectal cancer

    DEFF Research Database (Denmark)

    Nicoli, Elena Raluca; Dumitrescu, Theodor; Uscatu, Constantin Daniel;

    2014-01-01

    Autophagy has emerged not only as an essential repair mechanism to degrade damaged organelles and proteins but also as a major player in protection of tumor cells from multiple stresses. It was shown that autophagy gene polymorphisms are correlated with development of chronic inflammatory lesions...

  2. Protective Effects of Gastrodin Against Autophagy-Mediated Astrocyte Death.

    Science.gov (United States)

    Wang, Xin-Shang; Tian, Zhen; Zhang, Nan; Han, Jing; Guo, Hong-Liang; Zhao, Ming-Gao; Liu, Shui-Bing

    2016-03-01

    Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 μM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3-II, P62, and Beclin-1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl-2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 μM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin-1 expression. Furthermore, gastrodin blocked the down-regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26643508

  3. Characterization of a novel autophagy-specific gene, ATG29

    International Nuclear Information System (INIS)

    Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Δ cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins

  4. Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Xiao HAN; Jian-xun LIU; Xin-zhi LI

    2011-01-01

    Aim: To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes.Methods: Cardiac myocytes were incubated under starvation conditions (GD) for O, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sat B (50 μmol/L) in the presence or absence of chloro-quine (3 μmol/L) under GD 3 h, the amount of LC3-11, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. More-over, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis.Results: Immunoblot analysis showed that the amount of LC3-11 in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-11, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present.Conclusion: Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy.

  5. Polymorphisms in autophagy genes and susceptibility to tuberculosis.

    Directory of Open Access Journals (Sweden)

    Mario Songane

    Full Text Available Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02 and MTOR (p = 0.02 and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04. All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.

  6. Gene expression profiles of autophagy-related genes in multiple sclerosis.

    Science.gov (United States)

    Igci, Mehri; Baysan, Mehmet; Yigiter, Remzi; Ulasli, Mustafa; Geyik, Sirma; Bayraktar, Recep; Bozgeyik, İbrahim; Bozgeyik, Esra; Bayram, Ali; Cakmak, Ecir Ali

    2016-08-15

    Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate<0.05). Thus, altered expression levels of several autophagy related genes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis. PMID:27125224

  7. Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

    OpenAIRE

    Yongke Lu; Cederbaum, Arthur I.

    2015-01-01

    Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current re...

  8. mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaojun [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China); Zhong, Xiaomin [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China); Tanyi, Janos L.; Shen, Jianfeng [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Xu, Congjian [Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China); Gao, Peng [Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China); Zheng, Tim M. [Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); DeMichele, Angela [Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); Zhang, Lin, E-mail: linzhang@mail.med.upenn.edu [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States)

    2013-02-15

    Highlights: ► Gene set enrichment analysis indicated mir-30d might regulate the autophagy pathway. ► mir-30d represses the expression of BECN1, BNIP3L, ATG12, ATG5 and ATG2. ► BECN1, BNIP3L, ATG12, ATG5 and ATG2 are direct targets of mir-30d. ► mir-30d inhibits autophagosome formation and LC3B-I conversion to LC3B-II. ► mir-30d regulates the autophagy process. -- Abstract: In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.

  9. mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells

    International Nuclear Information System (INIS)

    Highlights: ► Gene set enrichment analysis indicated mir-30d might regulate the autophagy pathway. ► mir-30d represses the expression of BECN1, BNIP3L, ATG12, ATG5 and ATG2. ► BECN1, BNIP3L, ATG12, ATG5 and ATG2 are direct targets of mir-30d. ► mir-30d inhibits autophagosome formation and LC3B-I conversion to LC3B-II. ► mir-30d regulates the autophagy process. -- Abstract: In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy

  10. Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

    International Nuclear Information System (INIS)

    Highlights: •We observed the cell viability and death subjected to H/R in H9c2 cardiomyocytes. •We observed the degree of autophagy subjected to H/R in H9c2 cardiomyocytes. •LA inhibited the degree of autophagy in parallel to the enhanced cell survival. •LA inhibited the autophagy in parallel to the decreased total cell death. •We concluded that LA protected cardiomyocytes against H/R by inhibiting autophagy. -- Abstract: Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy

  11. Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Xueming; Chen, Aihua, E-mail: aihuachen2012@sina.com; Yang, Pingzhen; Song, Xudong; Liu, Yingfeng; Li, Zhiliang; Wang, Xianbao; Wang, Lizi; Li, Yunpeng

    2013-11-29

    Highlights: •We observed the cell viability and death subjected to H/R in H9c2 cardiomyocytes. •We observed the degree of autophagy subjected to H/R in H9c2 cardiomyocytes. •LA inhibited the degree of autophagy in parallel to the enhanced cell survival. •LA inhibited the autophagy in parallel to the decreased total cell death. •We concluded that LA protected cardiomyocytes against H/R by inhibiting autophagy. -- Abstract: Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.

  12. Melatonin Protects N2a against Ischemia/Reperfusion Injury through Autophagy Enhancement

    Institute of Scientific and Technical Information of China (English)

    国艳春; 王剑飞; 王忠强; 杨易; 王西明; 段秋红

    2010-01-01

    Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury.Although melatonin is known as an effective antioxidant,the mechanism of the protection cannot be explained merely by antioxidation.This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy,since autophagy has been frequently suggested to play a crucial role in neuron survival.To find it out,an ischemia/...

  13. Protective autophagy antagonizes oxaliplatin-induced apoptosis in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Ling Xu; Xiu-Juan Qu; Yun-Peng Liu; Ying-Ying Xu; Jing Liu; Ke-Zuo Hou; Ye Zhang

    2011-01-01

    Oxaliplatin-based chemotherapy is used for treating gastric cancer. Autophagy has been extensively implicated in cancer cells; however, its function is not fully understood. Our study aimed to determine if oxaliplatin induce autophagy in gastric cancer MGC803 cells and to assess the effect of autophagy on apoptosis induced by oxaliplatin. MGC803 cells were cultured with oxaliplatin. Cell proliferation was measured using MTT assay, and apoptosis was determined by flow cytometry. Protein expression was detected by Western blot. Autophagy was observed using fluorescent microscopy. Our results showed that the rate of apoptosis was 9.73% and 16.36% when MGC803 cells were treated with 5 and 20 μg/mL oxaliplatin for 24 h, respectively. In addition, caspase activation and poly ADP-ribose polymerase (PARP)cleavage were detected. Furthermore, when MGC803 cells were treated with oxaliplatin for 24 h, an accumulation of punctate LC3 and an increase of LC3-Ⅱ protein were also detected, indicating the activation of autophagy. Phosphorylation of Akt and mTOR were inhibited by oxaliplatin. Compared to oxaliplatin alone, the combination of autophagy inhibitor chlorochine and oxaliplatin significantly enhanced the inhibition of cell proliferation and the induction of cell apoptosis. In conclusion, oxaliplatin-induced protective autophagy partially prevents apoptosis in gastric cancer MGC803 cells. The combination of autophagy inhibitor and oxaliplatin may be a new therapeutic option for gastric cancer.

  14. Erythropoietin protects epithelial cells from excessive autophagy and apoptosis in experimental neonatal necrotizing enterocolitis.

    Directory of Open Access Journals (Sweden)

    Yueyue Yu

    Full Text Available Neonatal necrotizing enterocolitis (NEC is a devastating gastrointestinal disease of preterm infants. Increased intestinal epithelium permeability is an early event in NEC pathogenesis. Autophagy and apoptosis are induced by multiple stress pathways which may impact the intestinal barrier, and they have been associated with pathogenesis of diverse gastrointestinal diseases including inflammatory bowel disease. Using both in vitro and in vivo models, this study investigates autophagy and apoptosis under experimental NEC stresses. Furthermore this study evaluates the effect of erythropoietin (Epo, a component of breast milk previously shown to decrease the incidence of NEC and to preserve intestinal barrier function, on intestinal autophagy and apoptosis. It was found that autophagy and apoptosis are both rapidly up regulated in NEC in vivo as indicated by increased expression of the autophagy markers Beclin 1 and LC3II, and by evidence of apoptosis by TUNEL and cleaved caspase-3 staining. In the rat NEC experimental model, autophagy preceded the onset of apoptosis in intestine. In vitro studies suggested that Epo supplementation significantly decreased both autophagy and apoptosis via the Akt/mTOR signaling pathway and the MAPK/ERK pathway respectively. These results suggest that Epo protects intestinal epithelium from excessive autophagy and apoptosis in experimental NEC.

  15. Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Yongke Lu

    2015-10-01

    Full Text Available Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT, CYP2E1 knockout (KO or CYP2E1 humanized transgenic knockin (KI, mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA, an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These

  16. Targeting Protective Autophagy Exacerbates UV-Triggered Apoptotic Cell Death

    Directory of Open Access Journals (Sweden)

    Shih-Hwa Chiou

    2012-01-01

    Full Text Available Autophagy is activated by various stresses, including DNA damage, and previous studies of DNA damage-induced autophagy have focused on the response to chemotherapeutic drugs, ionizing radiation, and reactive oxygen species. In this study, we investigated the biological significance of autophagic response to ultraviolet (UV irradiation in A549 and H1299 cells. Our results indicated that UV induces on-rate autophagic flux in these cells. Autophagy inhibition resulting from the knockdown of beclin-1 and Atg5 reduced cell viability and enhanced apoptosis. Moreover, we found that ATR phosphorylation was accompanied by microtubule-associated protein 1 light chain 3B II (LC3B-II expression during the early phases following UV irradiation, which is a well-established inducer of ATR. Knocking down ATR further attenuated the reduction in LC3B-II at early stages in response to UV treatment. Despite the potential role of ATR in autophagic response, reduced ATR expression does not affect autophagy induction during late phases (24 and 48 h after UV treatment. The result is consistent with the reduced ATR phosphorylation at the same time points and suggests that autophagic response at this stage is activated via a distinct pathway. In conclusion, this study demonstrated that autophagy acts as a cytoprotective mechanism against UV-induced apoptosis and that autophagy induction accompanied with apoptosis at late stages is independent of ATR activation.

  17. Sinomenine Hydrochloride Protects against Polymicrobial Sepsis via Autophagy

    Directory of Open Access Journals (Sweden)

    Yu Jiang

    2015-01-01

    Full Text Available Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs. The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl is widely used to treat rheumatoid arthritis (RA. However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3 puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM. 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.

  18. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

    Directory of Open Access Journals (Sweden)

    Mengqiang Yu

    2014-10-01

    Full Text Available DNA damage-regulated autophagy modulator protein 1 (DRAM1, a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53 target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3 construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.

  19. Cannabinoid-induced autophagy: Protective or death role?

    Science.gov (United States)

    Costa, Lia; Amaral, Cristina; Teixeira, Natércia; Correia-da-Silva, Georgina; Fonseca, Bruno M

    2016-01-01

    Autophagy, the "self-digestion" mechanism of the cells, is an evolutionary conserved catabolic process that targets portions of cytoplasm, damaged organelles and proteins for lysosomal degradation, which plays a crucial role in development and disease. Cannabinoids are active compounds of Cannabis sativa and the most prevalent psychoactive substance is Δ(9)-tetrahydrocannabinol (THC). Cannabinoid compounds can be divided in three types: the plant-derived natural products (phytocannabinoids), the cannabinoids produced endogenously (endocannabinoids) and the synthesized compounds (synthetic cannabinoids). Various studies reported a cannabinoid-induced autophagy mechanism in cancer and non-cancer cells. In this review we focus on the recent advances in the cannabinoid-induced autophagy and highlight the molecular mechanisms involved in these processes. PMID:26732541

  20. Induction of cyto-protective autophagy by paramontroseite VO2 nanocrystals

    International Nuclear Information System (INIS)

    A variety of inorganic nanomaterials have been shown to induce autophagy, a cellular degradation process critical for the maintenance of cellular homeostasis. The overwhelming majority of autophagic responses elicited by nanomaterials were detrimental to cell fate and contributed to increased cell death. A widely held view is that the inorganic nanoparticles, when encapsulated and trapped by autophagosomes, may compromise the normal autophagic process due to the inability of the cells to degrade these materials and thus they manifest a detrimental effect on the well-being of a cell. Here we show that, contrary to this notion, nano-sized paramontroseite VO2 nanocrystals (P–VO2) induced cyto-protective, rather than death-promoting, autophagy in cultured HeLa cells. P–VO2 also caused up-regulation of heme oxygenase-1 (HO-1), a cellular protein with a demonstrated role in protecting cells against death under stress situations. The autophagy inhibitor 3-methyladenine significantly inhibited HO-1 up-regulation and increased the rate of cell death in cells treated with P–VO2, while the HO-1 inhibitor protoporphyrin IX zinc (II) (ZnPP) enhanced the occurrence of cell death in the P–VO2-treated cells while having no effect on the autophagic response induced by P–VO2. On the other hand, Y2O3 nanocrystals, a control nanomaterial, induced death-promoting autophagy without affecting the level of expression of HO-1, and the pro-death effect of the autophagy induced by Y2O3. Our results represent the first report on a novel nanomaterial-induced cyto-protective autophagy, probably through up-regulation of HO-1, and may point to new possibilities for exploiting nanomaterial-induced autophagy for therapeutic applications. (paper)

  1. Induction of cyto-protective autophagy by paramontroseite VO2 nanocrystals

    Science.gov (United States)

    Zhou, Wei; Miao, Yanyan; Zhang, Yunjiao; Liu, Liang; Lin, Jun; Yang, James Y.; Xie, Yi; Wen, Longping

    2013-04-01

    A variety of inorganic nanomaterials have been shown to induce autophagy, a cellular degradation process critical for the maintenance of cellular homeostasis. The overwhelming majority of autophagic responses elicited by nanomaterials were detrimental to cell fate and contributed to increased cell death. A widely held view is that the inorganic nanoparticles, when encapsulated and trapped by autophagosomes, may compromise the normal autophagic process due to the inability of the cells to degrade these materials and thus they manifest a detrimental effect on the well-being of a cell. Here we show that, contrary to this notion, nano-sized paramontroseite VO2 nanocrystals (P-VO2) induced cyto-protective, rather than death-promoting, autophagy in cultured HeLa cells. P-VO2 also caused up-regulation of heme oxygenase-1 (HO-1), a cellular protein with a demonstrated role in protecting cells against death under stress situations. The autophagy inhibitor 3-methyladenine significantly inhibited HO-1 up-regulation and increased the rate of cell death in cells treated with P-VO2, while the HO-1 inhibitor protoporphyrin IX zinc (II) (ZnPP) enhanced the occurrence of cell death in the P-VO2-treated cells while having no effect on the autophagic response induced by P-VO2. On the other hand, Y2O3 nanocrystals, a control nanomaterial, induced death-promoting autophagy without affecting the level of expression of HO-1, and the pro-death effect of the autophagy induced by Y2O3. Our results represent the first report on a novel nanomaterial-induced cyto-protective autophagy, probably through up-regulation of HO-1, and may point to new possibilities for exploiting nanomaterial-induced autophagy for therapeutic applications.

  2. Posttraumatic administration of luteolin protects mice from traumatic brain injury: implication of autophagy and inflammation.

    Science.gov (United States)

    Xu, Jianguo; Wang, Handong; Lu, Xinyu; Ding, Ke; Zhang, Li; He, Jin; Wei, Wuting; Wu, Yong

    2014-09-25

    Secondary brain insult induced by traumatic brain injury (TBI), including excitotoxicity, oxidative stress, inflammatory response, and neuronal degeneration, is sensitive to therapeutic interventions; therefore, searching for neuroprotective agents represents a promising therapeutic strategy for TBI treatment. Luteolin, a member of the flavonoid family, has recently been proven to modulate autophagy. However, whether it activates autophagy after TBI thereby alleviating the secondary insult is not yet understood. Here, we aimed to evaluate the neuroprotection of luteolin against TBI and the potential role of autophagy where it is involved. For this purpose, mice were randomly divided into four groups and then subjected to TBI. The treatment mice received luteolin at a dose of 30mg/kg 30min post-TBI based on our previous study. We employed western blot, immunofluorescence and quantitative real-time PCR to determine autophagy process and inflammatory response among different groups. Autophagy was found to be enhanced after luteolin treatment according to the expressions of autophagic markers. Furthermore, luteolin decreased nuclear accumulation of p65 induced by TBI, indicating attenuation of inflammation. In line with these observations, luteolin decreased mRNA and protein expressions of pro-inflammatory factors IL-1b and TNF-a. At last, luteolin reduced neuronal degeneration, and alleviated brain edema and blood-brain barrier (BBB) disruption. In conclusion, these results implied that luteolin protected mice brain from traumatic brain injury by inhibiting inflammatory response, and luteolin-induced autophagy might play a pivotal role in its neuroprotection. PMID:25093609

  3. Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Chenglong [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Zheng, Haining [Department of Hyperbaric Oxygen, Nanjing General Hospital of Nanjing Military Command, Nanjing (China); Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Ding, Dafa, E-mail: dingdafa2004@aliyun.com [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Lu, Yibing, E-mail: luyibing2004@126.com [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China)

    2015-10-01

    Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24 h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy. - Highlights: • High glucose leads to increased autophagy in podocytes at an early stage. • The early autophagic response protects against high glucose-induced apoptosis. • Heme oxygenase-1 enhances autophagy and decreases high glucose -mediated apoptosis. • Heme oxygenase-1 induces autophagy through the activation of AMPK.

  4. Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

    International Nuclear Information System (INIS)

    Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24 h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy. - Highlights: • High glucose leads to increased autophagy in podocytes at an early stage. • The early autophagic response protects against high glucose-induced apoptosis. • Heme oxygenase-1 enhances autophagy and decreases high glucose -mediated apoptosis. • Heme oxygenase-1 induces autophagy through the activation of AMPK

  5. Autophagy is a protective response to ethanol neurotoxicity

    OpenAIRE

    Chen, Gang; Ke, Zunji; Xu, Mei; Liao, Mingjun; Wang, Xin; Qi, Yuanlin; Zhang,Tao; Frank, Jacqueline A.; Bower, Kimberly A.; Shi, Xianglin; Luo, Jia

    2012-01-01

    Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II...

  6. β-Elemene-induced autophagy protects human gastric cancer cells from undergoing apoptosis

    International Nuclear Information System (INIS)

    β-Elemene, a compound found in an herb used in traditional Chinese medicine, has shown promising anti-cancer effects against a broad spectrum of tumors. The mechanism by which β-elemene kills cells remains unclear. The aim of the present study is to investigate the anti-tumor effect of β-elemene on human gastric cancer cells and the molecular mechanism involved. β-Elemene inhibited the viability of human gastric cancer MGC803 and SGC7901 cells in a dose-dependent manner. The suppression of cell viability was due to the induction of apoptosis. A robust autophagy was observed in the cells treated with β-elemene; it was characterized by the increase of punctate LC3 dots, the cellular morphology, and the increased levels of LC3-II protein. Further study showed that β-elemene treatment up-regulated Atg5-Atg12 conjugated protein but had little effect on other autophagy-related proteins. PI3K/Akt/mTOR/p70S6K1 activity was inhibited by β-elemene. Knockdown of Beclin 1 with small interfering RNA, or co-treatment with the autophagy inhibitor, 3-methyladenine or chlorochine enhanced significantly the antitumor effects of β-elemene. Our data provides the first evidence that β-elemene induces protective autophagy and prevents human gastric cancer cells from undergoing apoptosis. A combination of β-elemene with autophagy inhibitor might thus be a useful therapeutic option for advanced gastric cancer

  7. Autophagy Constitutes a Protective Mechanism against Ethanol Toxicity in Mouse Astrocytes and Neurons.

    Science.gov (United States)

    Pla, Antoni; Pascual, María; Guerri, Consuelo

    2016-01-01

    Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects. PMID:27070930

  8. Elemene injection induced autophagy protects human hepatoma cancer cells from starvation and undergoing apoptosis.

    Science.gov (United States)

    Lin, Yan; Wang, Keming; Hu, Chunping; Lin, Lin; Qin, Shukui; Cai, Xueting

    2014-01-01

    Elemene, a compound found in an herb used in traditional Chinese medicine, has shown promising anticancer effects against a broad spectrum of tumors. In an in vivo experiment, we found that apatinib, a tyrosine kinase inhibitor that selectively inhibits VEGFR2, combined with elemene injection (Ele) for the treatment of H22 solid tumor in mice resulted in worse effectiveness than apatinib alone. Moreover, Ele could protect HepG2 cells from death induced by serum-free starvation. Further data on the mechanism study revealed that Ele induced protective autophagy and prevented human hepatoma cancer cells from undergoing apoptosis. Proapoptosis effect of Ele was enhanced when proautophagy effect was inhibited by hydroxychloroquine. Above all, Ele has the effect of protecting cancer cells from death either in apatinib induced nutrient deficient environment or in serum-free induced starvation. A combination of elemene injection with autophagy inhibitor might thus be a useful therapeutic option for hepatocellular carcinoma. PMID:25152762

  9. No evidence for activated autophagy in left ventricular myocardium at early reperfusion with protection by remote ischemic preconditioning in patients undergoing coronary artery bypass grafting.

    Directory of Open Access Journals (Sweden)

    Nilgün Gedik

    Full Text Available Remote ischemic preconditioning (RIPC by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG. Activation of signal transducer and activator of transcription 5 (STAT5 in left ventricular (LV myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.CABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation or placebo (cuff deflated before skin incision (n = 10/10. Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline and at early (5-10 min reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p = 0.002. Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5-12 (ATG5-12 complex, light chain 3 (LC3, parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.Signals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p = 0.02. STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (≥2.7-fold versus baseline, p≤0.05.Protection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.

  10. Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xu-Guang [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China); Department of Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Ji, Tian-Xing [Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Xia, Yong, E-mail: gysyxy@gmail.com [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China); Ma, Yue-Yun, E-mail: cmbmayy@fmmu.edu.cn [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China)

    2013-03-08

    Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells

  11. Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

    International Nuclear Information System (INIS)

    Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells

  12. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ya C Wu

    Full Text Available Hydrogen sulfide (H(2S is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC and a panel of colon cancer cell lines (HT-29, SW1116, HCT116 were exposed to H(2S at concentrations similar to those found in the human colon. H(2S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2S was accompanied by G(1-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip. Moreover, exposure to H(2S led to features characteristic of autophagy, including increased formation of LC3B(+ autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2S. Further mechanistic investigation revealed that H(2S stimulated the phosphorylation of AMP-activated protein kinase (AMPK and inhibited the phosphorylation of mammalian target of rapamycin (mTOR and S6 kinase. Inhibition of AMPK significantly reversed H(2S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

  13. Tissue distribution, gender- and genotype-dependent expression of autophagy-related genes in avian species.

    Directory of Open Access Journals (Sweden)

    Alissa Piekarski

    Full Text Available As a result of the genetic selection of broiler (meat-type breeders chickens for enhanced growth rate and lower feed conversion ratio, it has become necessary to restrict feed intake. When broilers are fed ad libitum, they would become obese and suffer from several health-related problems. A vital adaptation to starvation is autophagy, a self-eating mechanism for recycling cellular constituents. The autophagy pathway has witnessed dramatic growth in the last few years and extensively studied in yeast and mammals however, there is a paucity of information in avian (non-mammalian species. Here we characterized several genes involved in autophagosome initiation and elongation in Red Jungle fowl (Gallus gallus and Japanese quail (coturnix coturnix Japonica. Both complexes are ubiquitously expressed in chicken and quail tissues (liver, leg and breast muscle, brain, gizzard, intestine, heart, lung, kidney, adipose tissue, ovary and testis. Alignment analysis showed high similarity (50.7 to 91.5% between chicken autophagy-related genes and their mammalian orthologs. Phylogenetic analysis demonstrated that the evolutionary relationship between autophagy genes is consistent with the consensus view of vertebrate evolution. Interestingly, the expression of autophagy-related genes is tissue- and gender-dependent. Furthermore, using two experimental male quail lines divergently selected over 40 generations for low (resistant, R or high (sensitive, S stress response, we found that the expression of most studied genes are higher in R compared to S line. Together our results indicate that the autophagy pathway is a key molecular signature exhibited gender specific differences and likely plays an important role in response to stress in avian species.

  14. Viruses, Autophagy Genes, and Crohn’s Disease

    OpenAIRE

    Hubbard, Vanessa M.; Ken Cadwell

    2011-01-01

    The etiology of the intestinal disease Crohn’s disease involves genetic factors as well as ill-defined environmental agents. Several genetic variants linked to this disease are associated with autophagy, a process that is critical for proper responses to viral infections. While a role for viruses in this disease remains speculative, accumulating evidence indicate that this possibility requires serious consideration. In this review, we will examine the three-way relationship between viruses, a...

  15. A SIRT3/AMPK/autophagy network orchestrates the protective effects of trans-resveratrol in stressed peritoneal macrophages and RAW 264.7 macrophages.

    Science.gov (United States)

    Duan, Wen-Jun; Li, Yi-Fang; Liu, Fang-Lan; Deng, Jie; Wu, Yan-Ping; Yuan, Wei-Lin; Tsoi, Bun; Chen, Jun-Li; Wang, Qi; Cai, Shao-Hui; Kurihara, Hiroshi; He, Rong-Rong

    2016-06-01

    Resveratrol gains a great interest for its strong antioxidant properties, while the molecular mechanisms underlie the beneficial effects on psychosocial stress remain controversial. In this study, we demonstrated that resveratrol protected peritoneal macrophages and RAW 264.7 cells from stress-induced decrease in the total cell count, phagocytic capability, reactive oxygen species generation, monodansylcadaverine and mitochondrial membrane potential in stressed mice. Resveratrol promoted stress-induced autophagy in both models. Modulation of autophagy by rapamycin or 3-methyladenine regulated the protective effect of resveratrol, suggesting a role of autophagy in the protective mechanisms of resveratrol. The comparison studies revealed that distinct mechanisms were implicated in the protective effect of resveratrol and other antioxidants (vitamin C and edaravone). Resveratrol promoted autophagy via upregulating SIRT3 expression and phosphorylation of AMP-activated protein kinase (AMPK). Knockdown of SIRT3 resulted in decreased autophagy and abolished protective effect of resveratrol. SIRT1 was also involved in the protective mechanism of resveratrol, although its effect on autophagy was unnoticeable. Pharmacological manipulation of autophagy modulated the effects of resveratrol on SIRT3 and AMPK, revealing the engagement of a positive feedback loop. In sharp contrast, vitamin C and edaravone effectively protected macrophages from stress-induced cytotoxicity, accompanied by downregulated SIRT3 expression and AMPK phosphorylation, and decreased level of autophagy response. Taken together, we conclude that a SIRT3/AMPK/autophagy network orchestrates in the protective effect of resveratrol in macrophages. PMID:27021965

  16. Corresponding erdosteine changes autophagy genes expression in hippocampus on Rhinitis medicamentosa model

    Directory of Open Access Journals (Sweden)

    Dokuyucu Recep

    2015-01-01

    Full Text Available In our study, rats were subjected to Oxymetazoline hydrochloride treatment and Rhinitis medicamentosa (RM was formed and then autophagy gene expression levels were determined after the application of an antioxidant agent erdosteine (ED. The rats were divided into three groups; Group 1 was the control group. Group 2 (RM and group 3 (RM+ED rats received two spray puffs of 0.05% oxymetazoline into the nasal cavities three times daily for eight weeks. After determination of RM in the rats, the RM group were killed. The ED+RM group received 10 mg/kg of an ED suspension. At the end of seven days, these rats were also killed. All groups’ hippocampus tissues were obtained for the measurement of autophagy gene expressions. In rhinitis medicamentosa group Atg5, Atg7 and Atg10 gene expressions in the left hippocampus were reduced as compared to control group (p=0.01, p>0.05, p=0.01, respectively. Also, erdosteine treatments were restored mRNA expression of autophagy genes. In right hippocampus of rhinitis medicamentosa group, Atg5 and Atg10 gene expressions was found to be down-regulated as compared to control group (p>0.05, p<0.05, respectively. Both BECN1 and ULK genes expression were found to be reduced in left hippocampus of rhinitis medicamentosa group. Erdosteine applications was restored the expression of these genes (p=0.03, p=0.03, respectively. Additionally, in right hippocampus, Erdosteine application was restored the expression of ULK gene (p=0.01. This is the first report that evaluated the expression autophagy genes in RM rat models and the changes observed after erdosteine applications.

  17. Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

    Science.gov (United States)

    Brigger, D; Schläfli, A M; Garattini, E; Tschan, M P

    2015-01-01

    All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously. PMID:26313912

  18. Upregulation of autophagy-related gene-5 (ATG-5 is associated with chemoresistance in human gastric cancer.

    Directory of Open Access Journals (Sweden)

    Jie Ge

    Full Text Available Autophagy-related gene-5 (ATG-5 is one of the key regulators of autophagic cell death. It has been widely regarded as a protective molecular mechanism for tumor cells during the course of chemotherapy. In the present study, we investigated the expression pattern of ATG-5 and multidrug resistance-associated protein-1 (MRP-1 in 135 gastric cancers (GC patients who were treated with epirubicin, cisplatin and 5-FU adjuvant chemotherapy (ECF following surgical resection and explored their potential clinical significance. We found that both ATG-5 (77.78% and MRP-1 (79.26% were highly expressed in GC patients. ATG-5 expression was significantly associated with depth of wall invasion, TNM stages and distant metastasis of GC (P<0.05, whereas MRP-1 expression was significantly linked with tumor size, depth of wall invasion, lymph node metastasis, TNM stages and differentiation status (P<0.05. ATG-5 expression was positively correlated with MRP-1 (rp = 0.616, P<0.01. Increased expression of ATG-5 and MPR-1 was significantly correlated with poor overall survival (OS; P<0.01 and disease free survival (DFS; P<0.01 of our GC cohort. Furthermore, we demonstrated that ATG-5 was involved in drug resistant of GC cells, which was mainly through regulating autophagy. Our data suggest that upregulated expression of ATG-5, an important molecular feature of protective autophagy, is associated with chemoresistance in GC. Expression of ATG-5 and MRP-1 may be independent prognostic markers for GC treatment.

  19. The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia

    International Nuclear Information System (INIS)

    Background and purpose: Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. Material and methods: Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. Results: ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. Conclusions: We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome

  20. Corresponding erdosteine changes autophagy genes expression in hippocampus on Rhinitis medicamentosa model

    OpenAIRE

    Dokuyucu Recep; Gogebakan Bulent; Cevik Cengiz

    2015-01-01

    In our study, rats were subjected to Oxymetazoline hydrochloride treatment and Rhinitis medicamentosa (RM) was formed and then autophagy gene expression levels were determined after the application of an antioxidant agent erdosteine (ED). The rats were divided into three groups; Group 1 was the control group. Group 2 (RM) and group 3 (RM+ED) rats received two spray puffs of 0.05% oxymetazoline into the nasal cavities three times daily for eight weeks. After...

  1. Quercitrin treatment protects endothelial progenitor cells from oxidative damage via inducing autophagy through extracellular signal-regulated kinase.

    Science.gov (United States)

    Zhi, Kangkang; Li, Maoquan; Bai, Jun; Wu, Yongfa; Zhou, Sili; Zhang, Xiaoping; Qu, Lefeng

    2016-07-01

    Atherosclerosis is a disease resulting from impaired endothelial function, often caused by oxidant injury or inflammation. Endothelial progenitor cells (EPCs) play a critical role in repairing damaged endothelium and protecting against atherosclerosis. Quercitrin, a plant-derived flavonoid compound, displays antioxidant and anti-inflammatory activities. In this study, we showed that quercitrin treatment reduced the apoptosis of EPCs caused by oxidized low-density lipoprotein (ox-LDL) in a dose-dependent manner. Quercitrin improved tube formation, migration and adhesion of ox-LDL-treated EPCs. To determine the effect of quercitrin in vivo, EPCs treated with or without ox-LDL and quercitrin were locally injected into the ischemic hind limb muscle of nude mice. Those injected with EPCs treated with ox-LDL and quercitrin showed significantly increased local accumulation of EPCs, blood flow recovery and capillary density compared with the control and ox-LDL only groups. Furthermore, we showed that quercitrin enhanced autophagy and upregulated mitogen-activated protein kinase and ERK phosphorylation in a dose-dependent manner in vitro. Autophagy inhibitors, chloroquine and 3-methyladenine, abrogated quercitrin-enhanced autophagy caused by ox-LDL as evidenced by decreased numbers of branch points, migratory cells and adherent cells, and increased numbers of apoptotic cells. The ERK inhibitor PD98059 abrogated quercitrin-enhanced autophagy, as identified by decreased autophagosome formation and downregulated ERK phosphorylation. The inhibition of ERK did not affect the expression of Rac1, but enhanced phosphorylation of Akt. Quercitrin treatment also increased the expression of E-cadherin, and PD98059 abrogated the upregulation of E-cadherin induced by quercitrin. Our findings suggested that autophagy is a protective mechanism in EPCs exposed to oxidative damage. Quercitrin can promote autophagy through the activation of ERK and the ERK signaling pathway is therefore

  2. A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

    Directory of Open Access Journals (Sweden)

    Ahnn Joohong

    2010-01-01

    Full Text Available Abstract Background Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines. Results We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. Conclusions We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future

  3. Taurine protects against As2O3-induced autophagy in livers of rat offsprings through PPARγ pathway.

    Science.gov (United States)

    Bai, Jie; Yao, Xiaofeng; Jiang, Liping; Zhang, Qiaoting; Guan, Huai; Liu, Shuang; Wu, Wei; Qiu, Tianming; Gao, Ni; Yang, Lei; Yang, Guang; Sun, Xiance

    2016-01-01

    Chronic exposures to arsenic had been associated with metabolism diseases. Peroxisome proliferator-activated receptor gamma (PPARγ) was found in the liver, regulated metabolism. Here, we found that the expression of PPARγ was decreased, the generation of reactive oxygen species (ROS) and autophagy were increased after treatment with As2O3 in offsprings' livers. Taurine (Tau), a sulfur-containing β-amino acid could reverse As2O3-inhibited PPARγ. Tau also inhibit the generation of ROS and autophagy. We also found that As2O3 caused autophagic cell death and ROS accelerated in HepG2 cells. Before incubation with As2O3, the cells were pretreated with PPARγ activator Rosiglitazone (RGS), we found that autophagy and ROS was inhibited in HepG2 cells, suggesting that inhibition of PPARγ contributed to As2O3-induced autophagy and the generation of ROS. After pretreatment with Tau, the level of PPARγ was improved and the autophagy and ROS was inhibited in As2O3-treated cells, suggesting that Tau could protect hepatocytes against As2O3 through modulating PPARγ pathway. PMID:27291853

  4. Inhibition of glycogen synthase kinase 3β promotes autophagy to protect mice from acute liver failure mediated by peroxisome proliferator-activated receptor α

    OpenAIRE

    Ren, F.; Zhang, L; Zhang, X; Shi, H; T. Wen; Bai, L.; S. Zheng; Y. Chen; Chen, D.; Li, L.; Duan, Z

    2016-01-01

    Our previous studies have demonstrated that inhibition of glycogen synthase kinase 3β (GSK3β) activity protects mice from acute liver failure (ALF), whereas its protective and regulatory mechanism remains elusive. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of the present study was to test the hypothesis that inhibition of GSK3β mediates autophagy to inhibit liver inflammation and protect against ALF. In ALF mice model induced by d-gala...

  5. Autophagy protects monocytes from Wolbachia heat shock protein 60-induced apoptosis and senescence.

    Directory of Open Access Journals (Sweden)

    Vijayan Kamalakannan

    2015-04-01

    Full Text Available Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60 interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence.

  6. Autophagy protects monocytes from Wolbachia heat shock protein 60-induced apoptosis and senescence.

    Science.gov (United States)

    Kamalakannan, Vijayan; Shiny, Abijit; Babu, Subash; Narayanan, Rangarajan Badri

    2015-04-01

    Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence. PMID:25849993

  7. Autophagy Induction Protects Against 7-Oxysterol-induced Cell Death via Lysosomal Pathway and Oxidative Stress

    OpenAIRE

    Xi-Ming Yuan; Nargis Sultana; Nabeel Siraj; Ward, Liam J.; Bijar Ghafouri; Wei Li

    2016-01-01

    7-Oxysterols are major toxic components in oxidized low-density lipoprotein and human atheroma lesions, which cause lysosomal membrane permeabilization (LMP) and cell death. Autophagy may function as a survival mechanism in this process. Here, we investigated whether 7-oxysterols mixed in an atheroma-relevant proportion induce autophagy, whether autophagy induction influences 7-oxysterol-mediated cell death, and the underlying mechanisms, by focusing on cellular lipid levels, oxidative stress...

  8. Characterization of an Autophagy-related Gene MdATG8i from apple

    Directory of Open Access Journals (Sweden)

    Ping eWang

    2016-05-01

    Full Text Available Nutrient deficiencies restrict apple (Malus sp. tree growth and productivity in Northwest China. The process of autophagy, a conserved degradation pathway in eukaryotic cells, has important roles in nutrient-recycling and helps improve plant performance during periods of nutrient-starvation. Little is known about the functioning of autophagy-related genes (ATGs in apple. In this study, one of the ATG8 gene family members MdATG8i was isolated from M. domestica. MdATG8i has conserved putative tubulin binding sites and ATG7 interaction domains. A 1865-bp promoter region cloned from apple genome DNA was predicated to have cis-regulatory elements responsive to light, environmental stresses and hormones. MdATG8i transcriptions were induced in response to leaf senescence, nitrogen depletion, and oxidative stress. At cellular level, MdATG8i protein was expressed in the nucleus and cytoplasm of onion epidermal cells. Yeast two-hybrid tests showed that MdATG8i could interact with MdATG7a and MdATG7b. In Arabidopsis, its heterologous expression was associated with enhanced vegetative growth, leaf senescence, and tolerance to nitrogen- and carbon-starvation. MdATG8i-overexpressing ‘Orin’ apple callus lines also displayed improved tolerance to nutrient-limited conditions. Our results demonstrate that MdATG8i protein could function in autophagy in a conserved way, as a positive regulator in the response to nutrient-starvation.

  9. Autophagy in Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Alexander J. S. Choi

    2011-01-01

    Full Text Available Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. During starvation, autophagy exerts a homeostatic function that promotes cell survival by recycling metabolic precursors. Additionally, autophagy can interact with other vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms, and thereby potentially influence disease pathogenesis. Macrophages deficient in autophagic proteins display enhanced caspase-1-dependent proinflammatory cytokine production and the activation of the inflammasome. Autophagy provides a functional role in infectious diseases and sepsis by promoting intracellular bacterial clearance. Mutations in autophagy-related genes, leading to loss of autophagic function, have been implicated in the pathogenesis of Crohn's disease. Furthermore, autophagy-dependent mechanisms have been proposed in the pathogenesis of several pulmonary diseases that involve inflammation, including cystic fibrosis and pulmonary hypertension. Strategies aimed at modulating autophagy may lead to therapeutic interventions for diseases associated with inflammation.

  10. Autophagy Protects Against Senescence and Apoptosis via the RAS-Mitochondria in High-Glucose-Induced Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2014-04-01

    Full Text Available Backgrounds: Autophagy is an important process in the pathogenesis of diabetes and plays a critical role in maintaining cellular homeostasis. However, the autophagic response and its mechanism in diabetic vascular endothelium remain unclear. Methods and Results: We studied high-glucose-induced renin-angiotensin system (RAS-mitochondrial damage and its effect on endothelial cells. With regard to therapeutics, we investigated the beneficial effect of angiotensin-converting enzyme inhibitors (ACEIs or angiotensin II type 1 receptor blockers (ARBs against high-glucose-induced endothelial responses. High glucose activated RAS, enhanced mitochondrial damage and increased senescence, apoptosis and autophagic-responses in endothelial cells, and these effects were mimicked by using angiotensin II (Ang. The use of an ACEI or ARB, however, inhibited the negative effects of high glucose. Direct mitochondrial injury caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP resulted in similar negative effects of high glucose or Ang and abrogated the protective effects of an ACEI or ARB. Additionally, by impairing autophagy, high-glucose-induced senescence and apoptosis were accelerated and the ACEI- or ARB-mediated beneficial effects were abolished. Furthermore, increases in FragEL™ DNA Fragmentation (TUNEL-positive cells, β-galactosidase activation and the expression of autophagic biomarkers were revealed in diabetic patients and rats, and the treatment with an ACEI or ARB decreased these responses. Conclusions: These data suggest that autophagy protects against senescence and apoptosis via RAS-mitochondria in high-glucose-induced endothelial cells.

  11. ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy

    Directory of Open Access Journals (Sweden)

    Johnson Kimberly N

    2010-04-01

    Full Text Available Abstract Background Autophagy is characterized by the sequestration of cytoplasm and organelles into multimembrane vesicles and subsequent degradation by the cell's lysosomal system. It is linked to many physiological functions in human cells including stress response, protein degradation, organelle turnover, caspase-independent cell death and tumor suppression. Malignant transformation is frequently associated with deregulation of autophagy and several tumor suppressors can modulate autophagic processes. The tumor suppressor p53 can induce autophagy after metabolic or genotoxic stress through transcriptionally-dependent and -independent mechanisms. In this study we expand on the former mechanism by functionally characterizing a p53 family target gene, ISG20L1 under conditions of genotoxic stress. Results We identified a p53 target gene, ISG20L1, and show that transcription of the gene can be regulated by all three p53 family members (p53, p63, and p73. We generated an antibody to ISG20L1 and found that it localizes to the nucleolar and perinucleolar regions of the nucleus and its protein levels increase in a p53- and p73-dependent manner after various forms of genotoxic stress. When ectopically expressed in epithelial cancer-derived cell lines, ISG20L1 expression decreased clonogenic survival without a concomitant elevation in apoptosis and this effect was partially rescued in cells that were ATG5 deficient. Knockdown of ISG20L1 did not alter 5-FU induced apoptosis as assessed by PARP and caspase-3 cleavage, sub-G1 content, and DNA laddering. Thus, we investigated the role of ISG20L1 in autophagy, a process commonly associated with type II cell death, and found that ISG20L1 knockdown decreased levels of autophagic vacuoles and LC3-II after genotoxic stress as assessed by electron microscopy, biochemical, and immunohistochemical measurements of LC3-II. Conclusions Our identification of ISG20L1 as a p53 family target and discovery that modulation

  12. Taurine protects against As2O3-induced autophagy in pancreas of rat offsprings through Nrf2/Trx pathway.

    Science.gov (United States)

    Bai, Jie; Yao, Xiaofeng; Jiang, Liping; Qiu, Tianming; Liu, Shuang; Qi, Baoxu; Zheng, Yue; Kong, Yuan; Yang, Guang; Chen, Min; Liu, Xiaofang; Sun, Xiance

    2016-04-01

    Arsenic was increasingly to blame as a risk factor for type 2 diabetes mellitus. In our previous study, we had found iAs stimulated autophagic flux and caused autophagic cell death through ROS pathway in INS-1 cells. Since NF-E2-related factor 2 (Nrf2) and the thioredoxin (Trx) system was a crucial line of defense against ROS, we investigated whether Nrf2/Trx pathway contributed to As2O3-stimulated autophagy and the role of taurine in this study. After treatment with 2 mg/kg BW-8 mg/kg BW As2O3 for 57 d, the expression of Nrf2 protein was decreased significantly in offsprings' pancreas. The expression of Trx gene was decreased significantly in pancreas subsequently. Finally, the generation of reactive oxygen species stimulated autophagy in arsenic-treated pancreas. Taurine could reverse arsenic-inhibited Nrf2 and Trx and inhibit autophagy. In short, inhibition of Nrf2/Trx pathway might play an important role in the pathogenesis of arsenic-related diabetes. Taurine could serve as nutrition supplementation against arsenic-related diabetes in high arsenic exposure area. PMID:26775255

  13. Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation from Latency by Preventing Virus-Induced Systemic Inflammation.

    Science.gov (United States)

    Park, Sunmin; Buck, Michael D; Desai, Chandni; Zhang, Xin; Loginicheva, Ekaterina; Martinez, Jennifer; Freeman, Michael L; Saitoh, Tatsuya; Akira, Shizuo; Guan, Jun-Lin; He, You-Wen; Blackman, Marcia A; Handley, Scott A; Levine, Beth; Green, Douglas R; Reese, Tiffany A; Artyomov, Maxim N; Virgin, Herbert W

    2016-01-13

    Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency. PMID:26764599

  14. Autophagy in infection.

    Science.gov (United States)

    Deretic, Vojo

    2010-04-01

    Autophagy is a ubiquitous eukaryotic cytoplasmic quality and quantity control pathway. The role of autophagy in cytoplasmic homeostasis seamlessly extends to cell-autonomous defense against intracellular microbes. Recent studies also point to fully integrated, multitiered regulatory and effector connections between autophagy and nearly all facets of innate and adaptive immunity. Autophagy in the immune system as a whole confers measured immune responses; on the flip side, suppression of autophagy can lead to inflammation and tissue damage, as evidenced by Crohn's disease predisposition polymorphisms in autophagy basal apparatus (Atg16L) and regulatory (IRGM) genes. Polymorphisms in the IRGM gene in human populations have also been linked to predisposition to tuberculosis. There are several areas of most recent growth: first, links between autophagy regulators and infectious disease predisposition in human populations; second, demonstration of a role for autophagy in infection control in vivo in animal models; third, the definition of specific antiautophagic defenses in highly evolved pathogens; and fourth, recognition of connections between the ubiquitin system and autophagy of bacteria (and interestingly mitochondria, which are incidentally organelles of bacterial evolutionary origin) via a growing list of modifier and adapter proteins including p62/SQSTM1, NDP52, Atg32, Parkin, and Nix/BNIP3L. PMID:20116986

  15. Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats.

    Science.gov (United States)

    Fang, Lili; Li, Xue; Zhong, Yinbo; Yu, Jing; Yu, Lina; Dai, Haibin; Yan, Min

    2015-10-01

    Cerebral microvascular endothelial cells (ECs) are crucial for brain vascular repair and maintenance, but their physiological function may be impaired during ischemic stroke and diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, could exacerbate ischemia-induced EC injury and dysfunction. We investigated the protective effect of autophagy on cultured human brain microvascular endothelial cells (HBMEC) that underwent MGO treatment. A further study was conducted to explore the underlying mechanisms of the protective effect. Autophagic activity was assessed by evaluating protein levels, using western blot. 3-methyladenine (3-MA), bafilomycin A1, ammonium chloride (AC), Beclin 1 siRNA, and chloroquine (CQ) were used to cause autophagy inhibition. Alarmar blue assay and lactate dehydrogenase release assay were used to evaluate cell viability. Streptozotocin was administered to induce type I diabetes in rats and post-permanent middle cerebral artery occlusion was performed to elicit cerebral ischemia. Blood-brain barrier permeability was also assessed. Our study found that MGO reduced HBMEC cell viability in a concentration- and time-dependent manner, and triggered the responsive autophagy activation. Autophagy inhibitors bafilomycin A1, AC, 3-MA, and BECN1 siRNA exacerbated MGO-induced HBMEC injury. FAK phosphorylation inhibitor PF573228 inhibited MGO-triggered autophagy and enhanced lactate dehydrogenase release. Meanwhile, similar autophagy activation in brain vascular ECs was observed during permanent middle cerebral artery occlusion-induced cerebral ischemia in diabetic rats, while chloroquine-induced autophagy inhibition enhanced blood-brain barrier permeability. Taken together, our study indicates that autophagy triggered by MGO defends HBMEC against injuries. PMID:26251121

  16. Cationic poly(amidoamine) dendrimers induced cyto-protective autophagy in hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Poly(amidoamine) (PAMAM) dendrimers are proposed as one of the most promising nanomaterials for biomedical applications because of their unique tree-like structure, monodispersity and tunable properties. In this study, we found that PAMAM dendrimers could induce the formation of autophagosomes and the conversion of microtubule-associated protein 1 light chain 3 (LC3) in hepatocellular carcinoma HepG2 cells, while the inhibition of the Akt/mTOR and activation of the Erk 1/2 signaling pathways were involved in autophagy-induced by PAMAM dendrimers. We also investigated the suppression of autophagy with the obviously enhanced cytotoxicity of PAMAM dendrimers. Moreover, the blockage of a reactive oxygen species (ROS) could enhance the growth inhibition and apoptosis of hepatocellular carcinoma cells, induced by PAMAM dendrimers through reducing autophagic effects. Taken together, these findings explored the role and mechanism of autophagy induced by PAMAM dendrimers in HepG2 cells, provided new insight into the effect of autophagy on drug delivery nanomaterials and tumor cells and contributed to the use of a drug delivery vehicle for hepatocellular carcinoma treatment. (paper)

  17. Cationic poly(amidoamine) dendrimers induced cyto-protective autophagy in hepatocellular carcinoma cells

    Science.gov (United States)

    Li, Yubin; Wang, Shaofei; Wang, Ziyu; Qian, Xiaolu; Fan, Jiajun; Zeng, Xian; Sun, Yun; Song, Ping; Feng, Meiqing; Ju, Dianwen

    2014-09-01

    Poly(amidoamine) (PAMAM) dendrimers are proposed as one of the most promising nanomaterials for biomedical applications because of their unique tree-like structure, monodispersity and tunable properties. In this study, we found that PAMAM dendrimers could induce the formation of autophagosomes and the conversion of microtubule-associated protein 1 light chain 3 (LC3) in hepatocellular carcinoma HepG2 cells, while the inhibition of the Akt/mTOR and activation of the Erk 1/2 signaling pathways were involved in autophagy-induced by PAMAM dendrimers. We also investigated the suppression of autophagy with the obviously enhanced cytotoxicity of PAMAM dendrimers. Moreover, the blockage of a reactive oxygen species (ROS) could enhance the growth inhibition and apoptosis of hepatocellular carcinoma cells, induced by PAMAM dendrimers through reducing autophagic effects. Taken together, these findings explored the role and mechanism of autophagy induced by PAMAM dendrimers in HepG2 cells, provided new insight into the effect of autophagy on drug delivery nanomaterials and tumor cells and contributed to the use of a drug delivery vehicle for hepatocellular carcinoma treatment.

  18. Autophagy Plays a Protective Role in Tumor Necrosis Factor-α-Induced Apoptosis of Bone Marrow-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Yang, Rui; Ouyang, Yi; Li, Weiping; Wang, Peng; Deng, Haiquan; Song, Bin; Hou, Jingyi; Chen, Zhong; Xie, Zhongyu; Liu, Zhenhua; Li, Jinteng; Cen, Shuizhong; Wu, Yanfeng; Shen, Huiyong

    2016-05-15

    Bone marrow-derived mesenchymal stem cells (BMSCs) are being broadly investigated for treating numerous inflammatory diseases. However, the low survival rate of BMSCs during the transplantation process has limited their application. Autophagy can maintain cellular homeostasis and protect cells against environmental stresses. Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine that can induce both autophagy and apoptosis of BMSCs. However, the actual role of autophagy in TNF-α-induced apoptosis of BMSCs remains poorly understood. In the current study, BMSCs were treated with TNF-α/cycloheximide (CHX), and cell death was examined by the Cell Counting Kit-8, Hoechst 33342 staining, and flow cytometric analysis as well as by the level of caspase-3 and caspase-8. Meanwhile, autophagic flux was examined by analyzing the level of microtubule-associated protein light chain 3 B (LC3B)-II and SQSTEM1/p62 and by examining the amount of green fluorescent protein-LC3B by fluorescence microscopy. Then, the cell death and autophagic flux of BMSCs were examined after pretreatment and cotreatment with 3-methyladenine (3-MA, autophagy inhibitor) or rapamycin (Rap, autophagy activator) together with TNF-α/CHX. Moreover, BMSCs pretreated with lentiviruses encoding short hairpin RNA of beclin-1 (BECN1) were treated with TNF-α/CHX, and then cell death and autophagic flux were detected. We showed that BMSCs treated with TNF-α/CHX presented dramatically elevated autophagic flux and cell death. Furthermore, we showed that 3-MA and shBECN1 treatment accelerated TNF-α/CHX-induced apoptosis, but that Rap treatment ameliorated cell death. Our results demonstrate that autophagy protects BMSCs against TNF-α-induced apoptosis. Enhancing the autophagy of BMSCs may elevate cellular survival in an inflammatory microenvironment. PMID:26985709

  19. Moxibustion Activates Macrophage Autophagy and Protects Experimental Mice against Bacterial Infection

    OpenAIRE

    Xiaojuan Li; Guanhua Guo; Feng Shen; Lihong Kong; Fengxia Liang; Guojie Sun

    2014-01-01

    Moxibustion is one of main therapies in traditional Chinese medicine and uses heat stimulation on the body surface from the burning of moxa to release pain or treat diseases. Emerging studies have shown that moxibustion can generate therapeutic effects by activating a series of signaling pathways and neuroendocrine-immune activities. Here we show moxibustion promoted profound macrophage autophagy in experimental Kunming mice, with reduced Akt phosphorylation and activated eIF2α phosphorylatio...

  20. Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3.

    Science.gov (United States)

    Duan, Liang; Yi, Min; Chen, Juan; Li, Shengjin; Chen, Weixian

    2016-05-13

    Autophagy plays a crucial role in the progress of Mycobacterium tuberculosis (MTB) infection. Recently, MTB enhanced intracellular survival (EIS) protein was reported to be secreted from MTB cells and linked to the inhibition of autophagy and the intracellular persistence of the pathogen. Here, we investigated the mechanism of EIS-mediated inhibition of autophagy in a human phorbol myristate acetate (PMA)-treated THP-1 cell line as well as in murine macrophages. We confirmed that the presence of EIS led to the inhibition of rapamycin (Rapa)-induced autophagy, while IL-10 gene expression was increased and Akt/mTOR/p70S6K pathway was activated during the process. IL-10 gene silencing led to a significant recovery of EIS-mediated autophagy suppression and decreased activity of the Akt/mTOR/p70S6K pathway. IL-10 promoter activity was unaffected by EIS. Remarkably, EIS increased the acetylation level of histone H3 (Ac-H3), which binds to the SP1 and STAT3 region of the human IL-10 gene promoter sequence. Thus, EIS protein possibly increased IL-10 expression through the regulation of Ac-H3 of its promoter. Our data demonstrated that one possible mechanism of the MTB evasion of autophagy is that the EIS protein up-regulates IL-10 via Ac-H3 and thus activates Akt/mTOR/p70S6K pathway. PMID:27079235

  1. Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes, BxATG1 and BxATG8

    Directory of Open Access Journals (Sweden)

    Li-Na Deng

    2016-03-01

    Full Text Available The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH. BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.

  2. Animal genes identification and mTOR signaling reactivation in autophagy

    Institute of Scientific and Technical Information of China (English)

    Xuejun Jiang

    2010-01-01

    @@ Autophagy(self-eating)is a self-degradation process essential for survival,differentiation,development,and homeostasis.Conceiving that a process of cellular self-eating could be beneficial may appear bewildering.In its simplest form,however,autophagy is probably a single cell's adjustment to starvation; the cell is forced to break down part of its own reserves to keep alive until circumstances improve(Mizushima and Klionsky,2007).

  3. Autophagy studies in Bombyx mori

    Directory of Open Access Journals (Sweden)

    L Tian

    2015-03-01

    Full Text Available Autophagy, which is well conserved from yeast to mammals, plays essential roles in development and diseases. Using the domesticated silkworm, Bombyx mori, as a model insect, several reports on autophagy have been made recently. Autophagic features are observed in the midgut and fat body during the larval-pupal transition as well as the silk gland and ovarian nurse cells during the pupal stage. There are 14 autophagy related (Atg genes, including at least two transcript variants of Atg1, predicated in Bombyx. Expression of most Atg genes is consistent with the autophagy process in the fat body during the larval-pupal transition, and reduction of Atg1 expression by RNAi blocks this process. The molting hormone, 20-hydroxyecdysone (20E, and starvation induce autophagy in the fat body by upregulating Atg gene expression and blocking the PI3K-TORC1 pathway. Meanwhile, autophagy precedes apoptosis in the midgut and other larval tissues during the larval-pupal transition, while the detailed mechanism is not illustrated yet. We assume that there are at least four future directions about autophagy studies in Bombyx during the next years: (1 physiological functions of autophagy; (2 identification of new components involved in the autophagy process; (3 detailed molecular mechanism of autophagosome formation; (4 functional relationship between autophagy and apoptosis.

  4. Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

    Science.gov (United States)

    Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

    2016-01-01

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD. PMID:27240523

  5. Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

    Science.gov (United States)

    Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

    2016-01-01

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

  6. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

    International Nuclear Information System (INIS)

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis

  7. Autophagy in the Pathogenesis of Disease

    OpenAIRE

    Levine, Beth; Kroemer, Guido

    2008-01-01

    Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development, and homeostasis. Autophagy principally serves an adaptive role to protect organisms against diverse pathologies, including infections, cancer, neurodegeneration, aging, and heart disease. However, in certain experimental disease settings, the self-cannibalistic or, paradoxically, even the prosurvival functions of autophagy may be deleterious. This Review summarizes recent advances in und...

  8. Autophagy During Cardiac Stress: Joys and Frustrations of Autophagy

    Science.gov (United States)

    Gottlieb, Roberta A.; Mentzer, Robert M.

    2013-01-01

    The study of autophagy has been transformed by the cloning of most genes in the pathway and the introduction of GFP-LC3 as a reporter to allow visual assessment of autophagy. The field of cardiac biology is not alone in attempting to understand the implications of autophagy. The purpose of this review is to address some of the controversies and conundrums associated with the evolving studies of autophagy in the heart. Autophagy is a cellular process involving a complex orchestration of regulatory gene products as well as machinery for assembly, selective targeting, and degradation of autophagosomes and their contents. Our understanding of the role of autophagy in human disease is rapidly evolving as investigators examine the process in different tissues and different pathophysiological contexts. In the field of heart disease, autophagy has been examined in the settings of ischemia and reperfusion, preconditioning, cardiac hypertrophy, and heart failure. This review addresses the role of autophagy in cardioprotection, the balance of catabolism and anabolism, the concept of mitochondrial quality control, and the implications of impaired autophagic flux or frustrated autophagy. PMID:20148666

  9. Autophagy: for better or for worse

    OpenAIRE

    Wirawan, Ellen; Berghe, Tom Vanden; Lippens, Saskia; Agostinis, Patrizia; Vandenabeele, Peter

    2011-01-01

    Autophagy is a lysosomal degradation pathway that degrades damaged or superfluous cell components into basic biomolecules, which are then recycled back into the cytosol. In this respect, autophagy drives a flow of biomolecules in a continuous degradation-regeneration cycle. Autophagy is generally considered a pro-survival mechanism protecting cells under stress or poor nutrient conditions. Current research clearly shows that autophagy fulfills numerous functions in vital biological processes....

  10. Autophagy in plants and phytopathogens.

    Science.gov (United States)

    Yoshimoto, Kohki; Takano, Yoshitaka; Sakai, Yasuyoshi

    2010-04-01

    Plants and plant-associated microorganisms including phytopathogens have to adapt to drastic changes in environmental conditions. Because of their immobility, plants must cope with various types of environmental stresses such as starvation, oxidative stress, drought stress, and invasion by phytopathogens during their differentiation, development, and aging processes. Here we briefly describe the early studies of plant autophagy, summarize recent studies on the molecular functions of ATG genes, and speculate on the role of autophagy in plants and phytopathogens. Autophagy regulates senescence and pathogen-induced cell death in plants, and autophagy and pexophagy play critical roles in differentiation and the invasion of host cells by phytopathogenic fungi. PMID:20079356

  11. Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling.

    Directory of Open Access Journals (Sweden)

    Jiaojiao Pang

    Full Text Available The endoplasmic reticulum (ER plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH.ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs. Myocardial mechanical and intracellular Ca(2+ properties, ER stress, autophagy and associated cell signaling molecules were evaluated.ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca(2+ homeostasis, oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62, along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene.Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy.

  12. The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway.

    Science.gov (United States)

    Bento, Carla F; Ashkenazi, Avraham; Jimenez-Sanchez, Maria; Rubinsztein, David C

    2016-01-01

    Forms of Parkinson's disease (PD) are associated with lysosomal and autophagic dysfunction. ATP13A2, which is mutated in some types of early-onset Parkinsonism, has been suggested as a regulator of the autophagy-lysosome pathway. However, little is known about the ATP13A2 effectors and how they regulate this pathway. Here we show that ATP13A2 depletion negatively regulates another PD-associated gene (SYT11) at both transcriptional and post-translational levels. Decreased SYT11 transcription is controlled by a mechanism dependent on MYCBP2-induced ubiquitination of TSC2, which leads to mTORC1 activation and decreased TFEB-mediated transcription of SYT11, while increased protein turnover is regulated by SYT11 ubiquitination and degradation. Both mechanisms account for a decrease in the levels of SYT11, which, in turn, induces lysosomal dysfunction and impaired degradation of autophagosomes. Thus, we propose that ATP13A2 and SYT11 form a new functional network in the regulation of the autophagy-lysosome pathway, which is likely to contribute to forms of PD-associated neurodegeneration. PMID:27278822

  13. Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yangling; Luo, Peihua; Wang, Jincheng; Dai, Jiabin; Yang, Xiaochun; Wu, Honghai; Yang, Bo, E-mail: yang924@zju.edu.cn; He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn

    2014-01-15

    Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination. - Highlights: • Autophagy inhibition could be a potential for combretastatin A-4 antitumor efficacy. • The JNK-Bcl-2 pathway plays a critical role in CA-4-induced autophagy. • ABT-737 enhances CA-4 anticancer activity due to inhibition of autophagy.

  14. Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

    International Nuclear Information System (INIS)

    Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination. - Highlights: • Autophagy inhibition could be a potential for combretastatin A-4 antitumor efficacy. • The JNK-Bcl-2 pathway plays a critical role in CA-4-induced autophagy. • ABT-737 enhances CA-4 anticancer activity due to inhibition of autophagy

  15. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    International Nuclear Information System (INIS)

    Research highlights: → We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. → Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. → This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  16. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

    Directory of Open Access Journals (Sweden)

    Kaisa Kyöstilä

    2015-04-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136 in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  17. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Hui [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Zhuo, Liling [College of Life Science, Zaozhuang University, Zaozhuang, Shandong, 277160 (China); Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China)

    2015-04-17

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

  18. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    International Nuclear Information System (INIS)

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival

  19. Heme oxygenase‑1 protects H2O2‑insulted glomerular mesangial cells from excessive autophagy.

    Science.gov (United States)

    Xu, Jia; Li, Jinshun; Wang, Jinhua; Chi, Yanchun; Zhang, Kun; Cui, Rui

    2016-06-01

    Increasing evidence has demonstrated that the activation of heme oxygenase (HO)‑1 reduces autophagy stimulated by oxidative stress injury, in which the supraphysiological production of reactive oxygen species (ROS) is detected. However, the potential mechanism underlying this effect remains unclear. The present study aimed to investigate the function of HO‑1 activation in the regulation of autophagy in glomerular mesangial cells subjected to H2O2‑induced oxidative stress injury. The results demonstrated that the HO‑1 agonist, hemin, reduces the LC3 protein level, which was enhanced by H2O2 treatment. Furthermore, hemin‑activated HO‑1 may function as a regulator of oxidative stress‑induced autophagy in a dose‑dependent manner. Pharmacological activation of c‑Jun N‑terminal kinase (JNK) inhibited the effect of hemin, indicating that the JNK signaling pathway is associated with the mechanism of HO‑1 in impeding excessive autophagy. In addition to successfully alleviating H2O2‑induced oxidative stress and cellular apoptosis, hemin‑activated HO‑1 may provide cytoprotection against rapamycin, a specific autophagy agonist. The present result suggested the inhibitory action of HO‑1 in the avoidance of a potentially enhanced linkage between autophagy and apoptosis, particularly in the setting of excessive ROS. Therefore, enhancing the intracellular activity of HO‑1 may assist the crosstalk between oxidative stress, autophagy and apoptosis, and represent a novel therapeutic strategy for renal ischemic disease. PMID:27122182

  20. Neuronal Autophagy and Neurodevelopmental Disorders

    OpenAIRE

    Lee, Kyung-Min; Hwang, Su-Kyung; Lee, Jin-A

    2013-01-01

    Neurodevelopmental disorders include a wide range of diseases such as autism spectrum disorders and mental retardation. Mutations in several genes that regulate neural development and synapse function have been identified in neurodevelopmental disorders. Interestingly, some affected genes and pathways in these diseases are associated with the autophagy pathway. Autophagy is a complex, bulky degradative process that involves the sequestration of cellular proteins, RNA, lipids, and cellular org...

  1. Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

    International Nuclear Information System (INIS)

    Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly, combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation

  2. Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Xiaopeng; Du, Jie; Hua, Song; Zhang, Haowen; Gu, Cheng; Wang, Jie; Yang, Lei; Huang, Jianfeng; Yu, Jiahua, E-mail: yujiahua@suda.edu.cn; Liu, Fenju, E-mail: fangsh@suda.edu.cn

    2015-01-15

    Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly, combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation.

  3. MOLECULAR CHARACTERIZATION OF AUTOPHAGY-RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS.

    Science.gov (United States)

    Liu, Kai-Yu; Xia, Yu-Qian; Zhou, Jing; Chen, Zu-Wen; Lu, Dandan; Zhang, Ning-Zhao; Liu, Xu-Sheng; Ai, Hui; Zhou, Li-Lin

    2016-08-01

    Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry-SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl-HP cells and partially co-localized with BmATG6-GFP, but it almost has no significant co-localization with GFP-HaATG8. Then, the Western blot analysis demonstrated that GFP-SeATG5 conjugated with ATG12. Moreover, real-time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions. PMID:27226059

  4. Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

    2015-07-01

    Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. PMID:25727880

  5. Molecular mechanism and regulation of autophagy

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Zhong-qin LIANG; Zhen-lun GU; Zheng-hong QIN

    2005-01-01

    Autophagy is a major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles in eukaryotic cells. A large number of intracellular/extracellular stimuli, including amino acid starvation and invasion of microorganisms, are able to induce the autophagic response in cells. The discovery of the ATG genes in yeast has greatly advanced our understanding of the molecular mechanisms participating in autophagy and the genes involved in regulating the autophagic pathway. Many yeast genes have mammalian homologs,suggesting that the basic machinery for autophagy has been evolutionarily conserved along the eukaryotic phylum. The regulation of autophagy is a very complex process. Many signaling pathways, including target of rapamycin (TOR) or mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase-I (PI3K-I)/PKB, GTPases, calcium and protein synthesis all play important roles in regulating autophagy. The molecular mechanisms and regulation of autophagy are discussed in this review.

  6. Evaluation of the anti-inflammatory action of curcumin analog (DM1): Effect on iNOS and COX-2 gene expression and autophagy pathways.

    Science.gov (United States)

    Paulino, Niraldo; Paulino, Amarilis Scremin; Diniz, Susana N; de Mendonça, Sergio; Gonçalves, Ivair D; Faião Flores, Fernanda; Santos, Reginaldo Pereira; Rodrigues, Carina; Pardi, Paulo Celso; Quincoces Suarez, José Agustin

    2016-04-15

    This work describes the anti-inflammatory effect of the curcumin-analog compound, sodium 4-[5-(4-hydroxy-3-methoxyphenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate (DM1), and shows that DM1 modulates iNOS and COX-2 gene expression in cultured RAW 264.7 cells and induces autophagy on human melanoma cell line A375. PMID:27010501

  7. Inducing autophagy

    DEFF Research Database (Denmark)

    Harder, Lea M; Bunkenborg, Jakob; Andersen, Jens S.

    2014-01-01

    Autophagy is a lysosomal-mediated catabolic process, which through degradation of different cytoplasmic components aids in maintaining cellular homeostasis and survival during exposure to extra- or intracellular stresses. Ammonia is a potential toxic and stress-inducing byproduct of glutamine...... catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used...... as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites...

  8. MAP1LC3B overexpression protects against Hermansky-Pudlak syndrome type-1-induced defective autophagy in vitro.

    Science.gov (United States)

    Ahuja, Saket; Knudsen, Lars; Chillappagari, Shashi; Henneke, Ingrid; Ruppert, Clemens; Korfei, Martina; Gochuico, Bernadette R; Bellusci, Saverio; Seeger, Werner; Ochs, Matthias; Guenther, Andreas; Mahavadi, Poornima

    2016-03-15

    Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, and some patients with HPS develop pulmonary fibrosis, known as HPS-associated interstitial pneumonia (HPSIP). We have previously reported that HPSIP is associated with severe surfactant accumulation, lysosomal stress, and alveolar epithelial cell type II (AECII) apoptosis. Here, we hypothesized that defective autophagy might result in excessive lysosomal stress in HPSIP. Key autophagy proteins, including LC3B lipidation and p62, were increased in HPS1/2 mice lungs. Electron microscopy demonstrated a preferable binding of LC3B to the interior of lamellar bodies in the AECII of HPS1/2 mice, whereas in wild-type mice it was present on the limiting membrane in addition to the interior of the lamellar bodies. Similar observations were noted in human HPS1 lung sections. In vitro knockdown of HPS1 revealed increased LC3B lipidation and p62 accumulation, associated with an increase in proapoptotic caspases. Overexpression of LC3B decreased the HPS1 knockdown-induced p62 accumulation, whereas rapamycin treatment did not show the same effect. We conclude that loss of HPS1 protein results in impaired autophagy that is restored by exogenous LC3B and that defective autophagy might therefore play a critical role in the development and progression of HPSIP. PMID:26719147

  9. Autophagy-related gene 5 and Wnt5 signaling pathway requires differentiation of embryonic stem cells into odontoblast-like cells.

    Science.gov (United States)

    Ozeki, Nobuaki; Mogi, Makio; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Matsumoto, Toru; Nakata, Kazuhiko

    2016-02-01

    We previously confirmed a unique and unanticipated role for an α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and matrix metalloproteinase (MMP)-3-mediated signaling cascade, in driving the odontoblast-like differentiation of mouse embryonic stem (ES) cells in a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). To explore the early signaling cascade for odontoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and Wnt signaling by CS/BMP-4 mediated odontoblast differentiation. In a screening experiment, CS/BMP-4 increased the mRNA and protein levels of Atg5, Lrp5/Fzd9 (an Atg5 receptor), and Wnt5, but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, and Atg12, together with the amount of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg5 and Wnt5 individually suppressed the CS/BMP-4-induced increase in odontoblast differentiation. The odontoblastic phenotype, involving dentin matrix protein-1 and dentin sialophosphoprotein expression, decreased when autophagy was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade involving Atg5, Wnt5a, α2 integrin, Emmprin, and MMP-3. This cascade results in a potent increase in odontoblastic cell differentiation, indicating the unique involvement of Atg5, autophagy and Wnt5 signaling in CS/BMP-4-induced differentiation of ES cells into odontoblast-like cells, at a relatively early stage. PMID:26806855

  10. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury. PMID:26103523

  11. Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway

    Science.gov (United States)

    Yang, Xiao-Jun; Si, Ruo-Huang; Liang, Yu-He; Ma, Bing-Qiang; Jiang, Ze-Bin; Wang, Bin; Gao, Peng

    2016-01-01

    AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival. METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay. RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3′ untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P pylori in AGS cells. CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway. PMID:27099441

  12. Dysregulation of Autophagy, Mitophagy, and Apoptotic Genes in the Medial Temporal Lobe Cortex in an Ischemic Model of Alzheimer’s Disease

    Science.gov (United States)

    Ułamek-Kozioł, Marzena; Kocki, Janusz; Bogucka-Kocka, Anna; Petniak, Alicja; Gil-Kulik, Paulina; Januszewski, Sławomir; Bogucki, Jacek; Jabłoński, Mirosław; Furmaga-Jabłońska, Wanda; Brzozowska, Judyta; Czuczwar, Stanisław J.; Pluta, Ryszard

    2016-01-01

    Ischemic brain damage is a pathological incident that is often linked with medial temporal lobe cortex injury and finally its atrophy. Post-ischemic brain injury associates with poor prognosis since neurons of selectively vulnerable ischemic brain areas are disappearing by apoptotic program of neuronal death. Autophagy has been considered, after brain ischemia, as a guardian against neurodegeneration. Consequently, we have examined changes in autophagy (BECN 1), mitophagy (BNIP 3), and apoptotic (caspase 3) genes in the medial temporal lobe cortex with the use of quantitative reverse-transcriptase PCR following transient 10-min global brain ischemia in rats with survival 2, 7, and 30 days. The intense significant overexpression of BECN 1 gene was noted on the 2nd day, while on days 7–30 the expression of this gene was still upregulated. BNIP 3 gene was downregulated on the 2nd day, but on days 7–30 post-ischemia, there was a significant reverse tendency. Caspase 3 gene, associated with apoptotic neuronal death, was induced in the same way as BNIP 3 gene after brain ischemia. Thus, the demonstrated changes indicate that the considerable dysregulation of expression of BECN 1, BNIP 3, and caspase 3 genes may be connected with a response of neuronal cells in medial temporal lobe cortex to transient complete brain ischemia. PMID:27472881

  13. Oxidative stress-induced autophagy: Role in pulmonary toxicity

    International Nuclear Information System (INIS)

    Autophagy is an evolutionarily conserved catabolic process important in regulating the turnover of essential proteins and in elimination of damaged organelles and protein aggregates. Autophagy is observed in the lung in response to oxidative stress generated as a consequence of exposure to environmental toxicants. Whether autophagy plays role in promoting cell survival or cytotoxicity is unclear. In this article recent findings on oxidative stress-induced autophagy in the lung are reviewed; potential mechanisms initiating autophagy are also discussed. A better understanding of autophagy and its role in pulmonary toxicity may lead to the development of new strategies to treat lung injury associated with oxidative stress. - Highlights: • Exposure to pulmonary toxicants is associated with oxidative stress. • Oxidative stress is known to induce autophagy. • Autophagy is upregulated in the lung following exposure to pulmonary toxicants. • Autophagy may be protective or pathogenic

  14. Oxidative stress-induced autophagy: Role in pulmonary toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Malaviya, Rama [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2014-03-01

    Autophagy is an evolutionarily conserved catabolic process important in regulating the turnover of essential proteins and in elimination of damaged organelles and protein aggregates. Autophagy is observed in the lung in response to oxidative stress generated as a consequence of exposure to environmental toxicants. Whether autophagy plays role in promoting cell survival or cytotoxicity is unclear. In this article recent findings on oxidative stress-induced autophagy in the lung are reviewed; potential mechanisms initiating autophagy are also discussed. A better understanding of autophagy and its role in pulmonary toxicity may lead to the development of new strategies to treat lung injury associated with oxidative stress. - Highlights: • Exposure to pulmonary toxicants is associated with oxidative stress. • Oxidative stress is known to induce autophagy. • Autophagy is upregulated in the lung following exposure to pulmonary toxicants. • Autophagy may be protective or pathogenic.

  15. Erythropoietin Modulates Autophagy Signaling in the Developing Rat Brain in an In Vivo Model of Oxygen-Toxicity

    Directory of Open Access Journals (Sweden)

    Marco Sifringer

    2012-10-01

    Full Text Available Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo. Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3, LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.

  16. The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients

    DEFF Research Database (Denmark)

    Metzger, Silke; Walter, Carolin; Riess, Olaf;

    2013-01-01

    , we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second......The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently...... independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the "REGISTRY" cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a...

  17. Identification of Barley (Hordeum vulgare L. Autophagy Genes and Their Expression Levels during Leaf Senescence, Chronic Nitrogen Limitation and in Response to Dark Exposure

    Directory of Open Access Journals (Sweden)

    Liliana Avila-Ospina

    2016-02-01

    Full Text Available Barley is a cereal of primary importance for forage and human nutrition, and is a useful model for wheat. Autophagy genes first described in yeast have been subsequently isolated in mammals and Arabidopsis thaliana. In Arabidopsis and maize it was recently shown that autophagy machinery participates in nitrogen remobilization for grain filling. In rice, autophagy is also important for nitrogen recycling at the vegetative stage. In this study, HvATGs, HvNBR1 and HvATI1 sequences were identified from bacterial artificial chromosome (BAC, complementary DNA (cDNA and expressed sequence tag (EST libraries. The gene models were subsequently determined from alignments between genome and transcript sequences. Essential amino acids were identified from the protein sequences in order to estimate their functionality. A total of twenty-four barley HvATG genes, one HvNBR1 gene and one HvATI1 gene were identified. Except for HvATG5, all the genomic sequences found completely matched their cDNA sequences. The HvATG5 gene sequence presents a gap that cannot be sequenced due to its high GC content. The HvATG5 coding DNA sequence (CDS, when over-expressed in the Arabidopsis atg5 mutant, complemented the plant phenotype. The HvATG transcript levels were increased globally by leaf senescence, nitrogen starvation and dark-treatment. The induction of HvATG5 during senescence was mainly observed in the flag leaves, while it remained surprisingly stable in the seedling leaves, irrespective of the leaf age during stress treatment.

  18. Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24.

    Directory of Open Access Journals (Sweden)

    Caryline Agler

    2014-02-01

    Full Text Available Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.

  19. Minocycline protects retinal ganglion cells after optic nerve crush injury in mice by delaying autophagy and upregulating nuclear factor-κB2

    Institute of Scientific and Technical Information of China (English)

    Jiao Xiaoling; Peng Yuan; Yang Liu

    2014-01-01

    Background Currently,no medicine is available that can prevent or treat neural damage associated with optic nerve injury.Minocycline is recently reported to have a neuroprotective function.The aims of this study were to exarmine the neuroprotective effect of minocycline on retinal ganglion cells (RGCs) and determine its underlying mechanisms,using a mouse model of optic nerve crush (ONC).Methods ONC was performed in the left eye of adult male mice,and the mice were randomly divided into minocycline-treated group and saline-treated control group.The mice without receiving ONC injury were used as positive controls.RGC densities were assessed in retinal whole mounts with immunofluorescence labeling of βⅢ-tubulin.Transmission electron microscopy was used to detect RGC morphologies,and Western blotting and real-time PCR were applied to investigate the expression of autophagy markers LC3-Ⅰ,LC3-Ⅱ,and transcriptional factors nuclear factor-κB1 (NF-κB1),NF-κB2.Results In the early stage after ONC (at Days 4 and 7),the density of RGCs in the minocycline-treated group was higher than that of the saline-treated group.Electron micrographs showed that minocycline prevented nuclei and mitochondria injuries at Day 4.Western blotting analysis demonstrated that the conversion of LC3-Ⅰ to LC3-Ⅱ was reduced in the minocycline-treated group at Days 4 and 7,which meant autophagy process was inhibited by minocycline.In addition,the gene expression of NF-κB2 was upregulated by minocycline at Day 4.Conclusion The neuroprotective effect of minocycline is generated in the early stage after ONC in mice,partly through delaying autophagy process and regulating NF-κB2 pathway.

  20. Modulating autophagy: a strategy for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Jun-Lin Li; Shao-Liang Han; Xia Fan

    2011-01-01

    Autophagy is a process in which long-lived proteins,damaged cell organelles,and other cellular particles are sequestered and degraded.This process is important for maintaining the cellular microenvironment when the cell is under stress.Many studies have shown that autophagy plays a complex role in human diseases,especially in cancer,where it is known to have paradoxical effects.Namely,autophagy provides the energy for metabolism and tumor growth and leads to cell death that promotes tumor suppression.The link between autophagy and cancer is also evident in that some of the genes that regulate carcinogenesis,oncogenes and tumor suppressor genes,participate in or impact the autophagy process.Therefore,modulating autophagy will be a valuable topic for cancer therapy.Many studies have shown that autophagy can inhibit the tumor growth when autophagy modulators are combined with radiotherapy and/or chemotherapy.These findings suggest that autophagy may be a potent target for cancer therapy.

  1. Mechanism of action of the tuberculosis and Crohn disease risk factor IRGM in autophagy.

    Science.gov (United States)

    Chauhan, Santosh; Mandell, Michael A; Deretic, Vojo

    2016-02-01

    Polymorphisms in the IRGM gene, associated with Crohn disease (CD) and tuberculosis, are among the earliest identified examples documenting the role of autophagy in human disease. Functional studies have shown that IRGM protects against these diseases by modulating autophagy, yet the exact molecular mechanism of IRGM's activity has remained unknown. We have recently elucidated IRGM's mechanism of action. IRGM functions as a platform for assembling, stabilizing, and activating the core autophagic machinery, while at the same time physically coupling it to conventional innate immunity receptors. Exposure to microbial products or bacterial invasion increases IRGM expression, which leads to stabilization of AMPK. Specific protein-protein interactions and post-translational modifications such as ubiquitination of IRGM, lead to a co-assembly with IRGM of the key autophagy regulators ULK1 and BECN1 in their activated forms. IRGM physically interacts with 2 other CD risk factors, ATG16L1 and NOD2, placing these 3 principal players in CD within the same molecular complex. This explains how polymorphisms altering expression or function of any of the 3 factors individually can affect the same process-autophagy. Furthermore, IRGM's interaction with NOD2, and additional pattern recognition receptors such as NOD1, RIG-I, and select TLRs, transduces microbial signals to the core autophagy apparatus. This work solves the long-standing enigma of how IRGM controls autophagy. PMID:26313894

  2. FGF signalling regulates bone growth through autophagy.

    Science.gov (United States)

    Cinque, Laura; Forrester, Alison; Bartolomeo, Rosa; Svelto, Maria; Venditti, Rossella; Montefusco, Sandro; Polishchuk, Elena; Nusco, Edoardo; Rossi, Antonio; Medina, Diego L; Polishchuk, Roman; De Matteis, Maria Antonietta; Settembre, Carmine

    2015-12-10

    Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes. PMID:26595272

  3. The role and modulation of autophagy in experimental models of myocardial ischemia-reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Carol Chen-Scarabelli; Richard Knight; Pratik R Agrawal; Louis Saravolatz; Cadigia Abuniat; Gabriele Scarabelli; Anastasis Stephanou; Leena Loomba; Jagat Narula; Tiziano M Scarabelli

    2014-01-01

    A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cyto-plasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion (I/R) injury. This tightly orchestrated cata-bolic cellular‘housekeeping’ process provides cells with a new source of energy to adapt to stressful conditions. This process was first described as a pro-survival mechanism, but increasing evidence suggests that it can also lead to the demise of the cell. Autophagy has been implicated in the pathogenesis of multiple cardiac conditions including myocardial I/R injury. However, a debate persists as to whether autophagy acts as a protec-tive mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the va-riability in the experimental models and species, and the methodology used to assess autophagy. This review provides updated knowledge on the modulation and role of autophagy in isolated cardiac cells subjected to I/R, and the growing interest towards manipulating autophagy to increase the survival of cardiac myocytes under conditions of stress-most notably being I/R injury. Perturbation of this evolutionarily conserved intracellular cleansing autophagy mechanism, by targeted modulation through, among others, mammalian target of rapamycin (mTOR) inhibitors, adenosine monophosphate-activated protein kinase (AMPK) modulators, calcium lowering agents, resveratrol, longevinex, sirtuin activators, the proapoptotic gene Bnip3, IP3 and lysosome inhibitors, may confer resistance to heart cells against I/R induced cell death. Thus, therapeutic ma-nipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling.

  4. Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.

    Directory of Open Access Journals (Sweden)

    Zhi-Hua Chen

    Full Text Available BACKGROUND: Chronic obstructive pulmonary disease (COPD is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS: Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7. Cigarette smoke extract (CSE is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1 and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1(-/- mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema. CONCLUSIONS: We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.

  5. The Scope of Gene Patent Protection and the TRIPS Agreement

    DEFF Research Database (Denmark)

    Sommer, Tine

    2007-01-01

    The Scope of Gene Patent Protection and the TRIPS Agreement - An Exclusively Nondiscriminatory Approach?   Gene patenting in Europe has provoked much debate both before and since the adoption of Directive 98/44/EC on the legal protection of biotechnological inventions. Some of the major points of...

  6. NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Sobhakumari, Arya [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Schickling, Brandon M. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Love-Homan, Laurie; Raeburn, Ayanna [Department of Pathology, The University of Iowa, Iowa City, IA (United States); Fletcher, Elise V.M. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Case, Adam J. [Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Domann, Frederick E. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); Miller, Francis J. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); and others

    2013-11-01

    Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: • Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. • Inhibition of autophagy sensitized HNSCC cells to erlotinib. • Erlotinib increased NOX4 promoter and 3′UTR luciferase activity. • Manipulating NOX4 decreases or increases autophagy.

  7. NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

    International Nuclear Information System (INIS)

    Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: • Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. • Inhibition of autophagy sensitized HNSCC cells to erlotinib. • Erlotinib increased NOX4 promoter and 3′UTR luciferase activity. • Manipulating NOX4 decreases or increases autophagy

  8. Autophagy in response to photodynamic therapy: cell survival vs. cell death

    Science.gov (United States)

    Oleinick, Nancy L.; Xue, Liang-yan; Chiu, Song-mao; Joseph, Sheeba

    2009-02-01

    Autophagy (or more properly, macroautophagy) is a pathway whereby damaged organelles or other cell components are encased in a double membrane, the autophagosome, which fuses with lysosomes for digestion by lysosomal hydrolases. This process can promote cell survival by removing damaged organelles, but when damage is extensive, it can also be a mechanism of cell death. Similar to the Kessel and Agostinis laboratories, we have reported the vigorous induction of autophagy by PDT; this was found in human breast cancer MCF-7 cells whether or not they were able to efficiently induce apoptosis. One way to evaluate the role of autophagy in PDT-treated cells is to silence one of the essential genes in the pathway. Kessel and Reiners silenced the Atg7 gene of murine leukemia L1210 cells using inhibitory RNA and found sensitization to PDT-induced cell death at a low dose of PDT, implying that autophagy is protective when PDT damage is modest. We have examined the role of autophagy in an epithelium-derived cancer cell by comparing parental and Atg7-silenced MCF-7 cells to varying doses of PDT with the phthalocyanine photosensitizer Pc 4. In contrast to L1210 cells, autophagy-deficient MCF-7 cells were more resistant to the lethal effects of PDT, as judged by clonogenic assays. A possible explanation for the difference in outcome for L1210 vs. MCF-7 cells is the greatly reduced ability of the latter to undergo apoptosis, a deficiency that may convert autophagy into a cell-death process even at low PDT doses. Experiments to investigate the mechanism(s) responsible are in process.

  9. Liver autophagy in anorexia nervosa and acute liver injury.

    Science.gov (United States)

    Kheloufi, Marouane; Boulanger, Chantal M; Durand, François; Rautou, Pierre-Emmanuel

    2014-01-01

    Autophagy, a lysosomal catabolic pathway for long-lived proteins and damaged organelles, is crucial for cell homeostasis, and survival under stressful conditions. During starvation, autophagy is induced in numerous organisms ranging from yeast to mammals, and promotes survival by supplying nutrients and energy. In the early neonatal period, when transplacental nutrients supply is interrupted, starvation-induced autophagy is crucial for neonates' survival. In adult animals, autophagy provides amino acids and participates in glucose metabolism following starvation. In patients with anorexia nervosa, autophagy appears initially protective, allowing cells to copes with nutrient deprivation. However, when starvation is critically prolonged and when body mass index reaches 13 kg/m(2) or lower, acute liver insufficiency occurs with features of autophagic cell death, which can be observed by electron microscopy analysis of liver biopsy samples. In acetaminophen overdose, a classic cause of severe liver injury, autophagy is induced as a protective mechanism. Pharmacological enhancement of autophagy protects against acetaminophen-induced necrosis. Autophagy is also activated as a rescue mechanism in response to Efavirenz-induced mitochondrial dysfunction. However, Efavirenz overdose blocks autophagy leading to liver cell death. In conclusion, in acute liver injury, autophagy appears as a protective mechanism that can be however blocked or overwhelmed. PMID:25250330

  10. Liver Autophagy in Anorexia Nervosa and Acute Liver Injury

    Directory of Open Access Journals (Sweden)

    Marouane Kheloufi

    2014-01-01

    Full Text Available Autophagy, a lysosomal catabolic pathway for long-lived proteins and damaged organelles, is crucial for cell homeostasis, and survival under stressful conditions. During starvation, autophagy is induced in numerous organisms ranging from yeast to mammals, and promotes survival by supplying nutrients and energy. In the early neonatal period, when transplacental nutrients supply is interrupted, starvation-induced autophagy is crucial for neonates’ survival. In adult animals, autophagy provides amino acids and participates in glucose metabolism following starvation. In patients with anorexia nervosa, autophagy appears initially protective, allowing cells to copes with nutrient deprivation. However, when starvation is critically prolonged and when body mass index reaches 13 kg/m2 or lower, acute liver insufficiency occurs with features of autophagic cell death, which can be observed by electron microscopy analysis of liver biopsy samples. In acetaminophen overdose, a classic cause of severe liver injury, autophagy is induced as a protective mechanism. Pharmacological enhancement of autophagy protects against acetaminophen-induced necrosis. Autophagy is also activated as a rescue mechanism in response to Efavirenz-induced mitochondrial dysfunction. However, Efavirenz overdose blocks autophagy leading to liver cell death. In conclusion, in acute liver injury, autophagy appears as a protective mechanism that can be however blocked or overwhelmed.

  11. Folate deprivation modulates the expression of autophagy- and circadian-related genes in HT-22 hippocampal neuron cells through GR-mediated pathway.

    Science.gov (United States)

    Sun, Qinwei; Yang, Yang; Li, Xi; He, Bin; Jia, Yimin; Zhang, Nana; Zhao, Ruqian

    2016-08-01

    Folic acid (FA) is an extremely important nutrient for brain formation and development. FA deficiency is highly linked to brain degeneration and age-related diseases, which are also associated with autophagic activities and circadian rhythm in hippocampal neurons. However, little is known how autophagy- and circadian-related genes in hippocampal neurons are regulated under FA deficiency. Here, hippocampal neuroncells (HT-22) were employed to determine the effect of FA deprivation (FD) on the expression of relevant genes and to reveal the potential role of glucocorticoid receptor (GR). FD increased autophagic activities in HT-22 cells, associated with significantly (PChIP assay showed that FD promoted (Pnetwork in response to folate deficiency. PMID:27133904

  12. Tumor Suppression and Promotion by Autophagy

    Directory of Open Access Journals (Sweden)

    Yenniffer Ávalos

    2014-01-01

    Full Text Available Autophagy is a highly regulated catabolic process that involves lysosomal degradation of proteins and organelles, mostly mitochondria, for the maintenance of cellular homeostasis and reduction of metabolic stress. Problems in the execution of this process are linked to different pathological conditions, such as neurodegeneration, aging, and cancer. Many of the proteins that regulate autophagy are either oncogenes or tumor suppressor proteins. Specifically, tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, Rheb, and AKT, inhibit autophagy, suggesting that autophagy is a tumor suppressor mechanism. Consistent with this hypothesis, the inhibition of autophagy promotes oxidative stress, genomic instability, and tumorigenesis. Nevertheless, autophagy also functions as a cytoprotective mechanism under stress conditions, including hypoxia and nutrient starvation, that promotes tumor growth and resistance to chemotherapy in established tumors. Here, in this brief review, we will focus the discussion on this ambiguous role of autophagy in the development and progression of cancer.

  13. The metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), inhibits stress-induced autophagy in cancer cells.

    Science.gov (United States)

    Sahni, Sumit; Bae, Dong-Hun; Lane, Darius J R; Kovacevic, Zaklina; Kalinowski, Danuta S; Jansson, Patric J; Richardson, Des R

    2014-04-01

    N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action. PMID:24532803

  14. Aggresome-autophagy involvement in a sarcopenic patient with rigid spine syndrome and a p.C150R mutation in FHL1 gene

    Directory of Open Access Journals (Sweden)

    Patrizia eSabatelli

    2014-08-01

    Full Text Available The four-and-half LIM domain protein 1 (FHL1 is highly expressed in skeletal and cardiac muscle. Mutations of the FHL1 gene have been associated with diverse chronic myopathies including reducing body myopathy (RBM, rigid spine syndrome, and Emery-Dreifuss muscular dystrophy. We investigated a family with a mutation (p.C150R in the second LIM domain of FHL1. In this family, a brother and a sister were affected by rigid spine syndrome, and their mother had a mild lower limbs weakness. The 34-year-old female had an early and progressive rigidity of the cervical spine and severe respiratory insufficiency. Muscle mass evaluated by DXA was markedly reduced, while fat mass was increased to 40%. CT scan showed an almost complete substitution of muscle by fibro-adipose tissue. Muscle biopsy showed accumulation of FHL1 throughout the cytoplasm and around myonuclei into multiproteic aggregates with aggresome/autophagy features as indicated by ubiquitin, p62 and LC3 labeling. DNA deposits, not associated with nuclear lamina components and histones, were also detected into the aggregates, suggesting nuclear degradation. Ultrastructural analysis showed the presence of dysmorphic nuclei, accumulation of tubulofilamentous and granular material and perinuclear accumulation of autophagic vacuoles. These data point to an involvement of the aggresome-autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation.

  15. Autophagy: A double-edged sword in intervertebral disk degeneration.

    Science.gov (United States)

    Zhang, Shu-Jun; Yang, Wei; Wang, Cheng; He, Wen-Si; Deng, Hai-Yang; Yan, Yi-Guo; Zhang, Jian; Xiang, Yong-Xiao; Wang, Wen-Jun

    2016-06-01

    Autophagy is a homeostatic mechanism through which intracellular damaged organelles and proteins are degraded and recycled in response to increased metabolic demands or stresses. Although primarily cytoprotective, dysfunction of autophagy is often associated with many degenerative diseases, including intervertebral disc (IVD) degeneration (IDD). As a main contributing factor to low back pain, IDD is the pathological basis for various debilitating spinal diseases. Either higher or lower levels of autophagy are observed in degenerative IVD cells. Despite the precise role of autophagy in disc degeneration that is still controversial, with difference from protection to aggravation, targeting autophagy has shown promise for mitigating disc degeneration. In the current review, we summarize the changes of autophagy in degenerative IVD cells and mainly discuss the relationship between autophagy and IDD. With continued efforts, modulation of the autophagic process could be a potential and attractive therapeutic strategy for degenerative disc disease. PMID:27018178

  16. Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

    International Nuclear Information System (INIS)

    Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G2/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21Waf1/Cip1. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B1, a cyclin required for progression through the G2/M phase. Taken together, DHA induces G2/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

  17. The role of autophagy in sensitizing malignant glioma cells to radiation therapy

    Institute of Scientific and Technical Information of China (English)

    Wenzhuo Zhuang; Zhenghong Qin; Zhongqin Liang

    2009-01-01

    Malignant gliomas representthe majority of primary brain tumors.The current standard treatments for malignant gliomas include surgical resection,radiation therapy,and chemotherapy.Radiotherapy,a standard adjuvant therapy,confers some survival advantages,but resistance of the glioma cells to the efficacy of radiation limits the success of the treatment.The mechanisms underlying glioma cell radioresistance have remained elusive.Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm.Recent studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy.Also,autophagy is a novel response of glioma cells to ionizing radiation.Autophagic cell death is considered programmed cell death type Ⅱ,whereas apoptosis is programmed cell death type Ⅰ.These two types of cell death are predominantly distinctive,but many studies demonstrate a cross-talk between them.Whether autophagy in cancer cells causes death or protects cells is controversial.The regulatory pathways of autophagy share several molecules.P13K/Akt/Mtor,DNA-PK,tumor suppressor genes, mitochondrial damage,and lysosome may play important roles in radiation-induced autophagy in glioma cells.Recently,a highly tumorigenic glioma tumor subpopulation,termed cancer stem cell or tumor-initiating cell,has been shown to promote therapeutic resistance.This review summarizes the main mediators associated with radiation-induced autophagy in malignant glioma cells and discusses the implications of the cancer stem cell hypothesis for the development of future therapies for brain tumors.

  18. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    International Nuclear Information System (INIS)

    Highlights: ► SIRT1 inactivation decreases autophagy in THP-1 cell. ► Inhibition of autophagy induces inflammation. ► SIRT1 inactivation induces inflammation through NF-κB activation. ► The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-κB activation. ► SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD+-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 through nuclear factor (NF)-κB signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-κB activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-κB activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and is implicated in decreased 5′-AMP activated kinase (AMPK) activation, leading to the impairment of autophagy. The mTOR inhibitor, rapamycin, abolishes

  19. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Koya, Daisuke, E-mail: koya0516@kanazawa-med.ac.jp [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer SIRT1 inactivation decreases autophagy in THP-1 cell. Black-Right-Pointing-Pointer Inhibition of autophagy induces inflammation. Black-Right-Pointing-Pointer SIRT1 inactivation induces inflammation through NF-{kappa}B activation. Black-Right-Pointing-Pointer The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-{kappa}B activation. Black-Right-Pointing-Pointer SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD{sup +}-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6 through nuclear factor (NF)-{kappa}B signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-{kappa}B activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-{kappa}B activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and

  20. Pyrvinium targets autophagy addiction to promote cancer cell death

    OpenAIRE

    Deng, Longfei; Lei, Yunlong; Liu, Rui; Li, Jingyi; Yuan, Kefei; Li, Yi; Chen, Yi; Liu, Yi; Lu, You; Edwards III, Carl K; Huang, Canhua; Wei, Yuquan

    2013-01-01

    Autophagy is a cellular catabolic process by which long-lived proteins and damaged organelles are degradated by lysosomes. Activation of autophagy is an important survival mechanism that protects cancer cells from various stresses, including anticancer agents. Recent studies indicate that pyrvinium pamoate, an FDA-approved antihelminthic drug, exhibits wide-ranging anticancer activity. Here we demonstrate that pyrvinium inhibits autophagy both in vitro and in vivo. We further demonstrate that...

  1. Hypoxia, MTOR and autophagy

    OpenAIRE

    Blagosklonny, Mikhail V.

    2013-01-01

    Although hypoxia can cause cell cycle arrest, it may simultaneously suppress a conversion from this arrest to senescence. Furthermore, hypoxia can suppress senescence caused by diverse stimuli, maintaining reversible quiescence instead. Hypoxia activates autophagy and inhibits MTOR, thus also activating autophagy. What is the relationship between autophagy and cellular senescence? Also, can inhibition of MTOR and stimulation of autophagy explain the gerosuppressive effects of hypoxia?

  2. Dexamethasone-induced autophagy mediates muscle atrophy through mitochondrial clearance

    OpenAIRE

    Troncoso, Rodrigo; Paredes, Felipe; Parra, Valentina; Gatica, Damián; Vásquez-Trincado, César; Quiroga, Clara; Bravo-Sagua, Roberto; López-Crisosto, Camila; Rodriguez, Andrea E; Oyarzún, Alejandra P; Kroemer, Guido; Lavandero, Sergio

    2014-01-01

    Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 a...

  3. WT1 is involved in the Akt-JNK pathway dependent autophagy through directly regulating Gas1 expression in human osteosarcoma cells.

    Science.gov (United States)

    Mo, Hao; He, Juliang; Yuan, Zhenchao; Mo, Ligen; Wu, Zhenjie; Lin, Xiang; Liu, Bin; Guan, Jian

    2016-09-01

    Macroautophagy (herein termed autophagy) works as a protective mechanism in tumorigenesis and development under metabolic stress condition. Multitudes of genes have been found involved in this process during past decades. In the present study, we report that Wilm's tumor suppressor1 (WT1) is involved in autophagy in osteosarcoma (OS) cells. WT1, a transcription factor with multitude of target genes, expresses in a majority of cancer types. Though wide-ranging effect of WT1 is now well documented, the function of WT1 in tumors remains poorly defined. In this chapter, it is found that high expression of WT1 positively correlates with active autophagy in human osteosarcoma cells. And further study on cell signaling pathway illustrates that Akt/JNK pathway acts as a positive regulator of autophagy induced by WT1. Here, we present evidence that WT1 modulates Akt/JNK signaling pathway mediated autophagy by controlling the expression of growth arrest-specific 1 (Gas1). We show that WT1 is required for Gas1 transcription in osteosarcoma cells. And Gas1 is upregulated followed WT1 overexpression in a time-dependent manner. Loss of Gas1 results in a reduction of WT1-induced autophagy. PMID:27453337

  4. Neferine Attenuates the Protein Level and Toxicity of Mutant Huntingtin in PC-12 Cells via Induction of Autophagy

    Directory of Open Access Journals (Sweden)

    Vincent Kam Wai Wong

    2015-02-01

    Full Text Available Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington’s disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74 in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  5. Protective Effect of Astaxanthin on Liver Fibrosis through Modulation of TGF-β1 Expression and Autophagy

    OpenAIRE

    Shen, Miao; CHEN, KAN; Lu, Jie; Cheng, Ping; Ling XU; Dai, Weiqi; Wang, Fan; He, Lei; Yan ZHANG; Chengfen, Wang; Li, Jingjing; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan

    2014-01-01

    Liver fibrosis is a common pathway leading to cirrhosis and a worldwide clinical issue. Astaxanthin is a red carotenoid pigment with antioxidant, anticancer, and anti-inflammatory properties. The aim of this study was to investigate the effect of astaxanthin on liver fibrosis and its potential protective mechanisms. Liver fibrosis was induced in a mouse model using CCL4 (intraperitoneal injection, three times a week for 8 weeks), and astaxanthin was administered everyday at three doses (20, 4...

  6. Role of autophagy in diabetes and endoplasmic reticulum stress of pancreatic β-cells

    OpenAIRE

    Quan, Wenying; Lim, Yu-Mi; Lee, Myung-Shik

    2012-01-01

    Type 2 diabetes mellitus is characterized by insulin resistance and failure of pancreatic β-cells producing insulin. Autophagy plays a crucial role in cellular homeostasis through degradation and recycling of organelles such as mitochondria or endoplasmic reticulum (ER). Here we discussed the role of β-cell autophagy in development of diabetes, based on our own studies using mice with β-cell-specific deletion of Atg7 (autophagy-related 7), an important autophagy gene, and studies by others. β...

  7. RUFY4: Immunity piggybacking on autophagy?

    Science.gov (United States)

    Terawaki, Seigo; Camosseto, Voahirana; Pierre, Philippe; Gatti, Evelina

    2016-03-01

    Although autophagy is a highly conserved mechanism among species and cell types, few are the molecules involved with the autophagic process that display cell- or tissue- specific expression. We have unraveled the positive regulatory role on autophagy of RUFY4 (RUN and FYVE domain containing 4), which is expressed in subsets of immune cells, including dendritic cells (DCs). DCs orchestrate the eradication of pathogens by coordinating the action of the different cell types involved in microbe recognition and destruction during the immune response. To fulfill this function, DC display particular regulation of their endocytic and autophagy pathways in response to the immune environment. Autophagy flux is downmodulated in DCs upon microbe sensing, but is remarkably augmented, when cells are differentiated in the presence of the pleiotropic cytokine IL4 (interleukin 4). From gene expression studies aimed at comparing the impact of IL4 on DC differentiation, we identified RUFY4, as a novel regulator that augments autophagy flux and, when overexpressed, induces drastic membrane redistribution and strongly tethers lysosomes. RUFY4 is therefore one of the few known positive regulators of autophagy that is expressed in a cell-specific manner or under specific immunological conditions associated with IL4 expression such as allergic asthma. PMID:26760128

  8. Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses

    OpenAIRE

    Kenzelmann Broz, Daniela; Spano Mello, Stephano; Bieging, Kathryn T.; Jiang, Dadi; Rachel L Dusek; Brady, Colleen A.; Sidow, Arend; Attardi, Laura D.

    2013-01-01

    To gain new insights into p53 biology, Kenzelmann Broz et al. used high-throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. This approach identified autophagy genes as direct p53 target genes. p53-induced autophagy was important for both p53-dependent apoptosis and transformation suppression by p53. These data highlight an intimate connection between p53 and autophagy and suggest that autophagy contributes to p53-...

  9. Tomato HsfA1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy

    OpenAIRE

    Wang, Yu; Cai, Shuyu; Yin, Lingling; Kai SHI; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Zhou, Jie

    2015-01-01

    Autophagy plays critical roles in plant responses to stress. In contrast to the wealth of information concerning the core process of plant autophagosome assembly, our understanding of the regulation of autophagy is limited. In this study, we demonstrated that transcription factor HsfA1a played a critical role in tomato tolerance to drought stress, in part through its positive role in induction of autophagy under drought stress. HsfA1a expression was induced by drought stress. Virus-induced Hs...

  10. Selective Autophagy in Drosophila

    Directory of Open Access Journals (Sweden)

    Ioannis P. Nezis

    2012-01-01

    Full Text Available Autophagy is an evolutionarily conserved process of cellular self-eating and is a major pathway for degradation of cytoplasmic material by the lysosomal machinery. Autophagy functions as a cellular response in nutrient starvation, but it is also associated with the removal of protein aggregates and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Although it was initially believed that autophagy occurs randomly in the cell, during the last years, there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective. Given the important role of autophagy and selective autophagy in several disease-related processes such as neurodegeneration, infections, and tumorigenesis, it is important to understand the molecular mechanisms of selective autophagy, especially at the organismal level. Drosophila is an excellent genetically modifiable model organism exhibiting high conservation in the autophagic machinery. However, the regulation and mechanisms of selective autophagy in Drosophila have been largely unexplored. In this paper, I will present an overview of the current knowledge about selective autophagy in Drosophila.

  11. The interplay between autophagy and ROS in tumorigenesis

    Directory of Open Access Journals (Sweden)

    VassilikiKarantza

    2012-11-01

    Full Text Available Reactive oxygen species (ROS at physiological levels are important cell signaling molecules. However, aberrantly high ROS are intimately associated with disease and commonly observed in cancer. Mitochondria are primary sources of intracellular ROS, and their maintenance is essential to cellular health. Autophagy, an evolutionarily conserved process whereby cytoplasmic components are delivered to lysosomes for degradation, is responsible for mitochondrial turnover and removal of damaged mitochondria. Impaired autophagy is implicated in many pathological conditions, including neurological disorders, inflammatory bowel disease, diabetes, aging and cancer. The first reports connecting autophagy to cancer showed that allelic loss of the essential autophagy gene BECLIN1 (BECN1 is prevalent in human breast, ovarian and prostate cancers and that Becn1+/- mice develop mammary gland hyperplasias, lymphomas, and lung and liver tumors. Subsequent studies demonstrated that Atg5-/- and Atg7-/- livers give rise to adenomas, Atg4-/- mice are susceptible to chemical carcinogenesis, and Bif1-/- mice are prone to spontaneous tumors, indicating that autophagy defects promote tumorigenesis. Due to defective mitophagy, autophagy-deficient cells accumulate damaged mitochondria and deregulated ROS levels, which likely contribute to their tumor-initiating capacity. However, the role of autophagy in tumorigenesis is complex, as more recent work also revealed tumor dependence on autophagy: autophagy-competent mutant-Ras-expressing cells form tumors more efficiently than their autophagy-deficient counterparts; similarly, FIP200 deficiency suppresses PyMT-driven mammary tumorigenesis. These latter findings are attributed to the fact that tumors driven by powerful oncogenes have high metabolic demands catered to by autophagy. In this review, we discuss the relationship between ROS and autophagy and summarize our current knowledge on their functional interactions in

  12. Chemical Inhibition of Autophagy

    DEFF Research Database (Denmark)

    Baek, Eric; Lin Kim, Che; Gyeom Kim, Mi;

    2016-01-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemic...

  13. Autophagy and cancer

    Institute of Scientific and Technical Information of China (English)

    Si-Zhao; Lu; Duygu; Dee; Harrison-Findik

    2013-01-01

    Autophagy is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles.Autophagy is activated in response to both physiological and pathological stimuli including growth factor depletion,energy deficiency or the upregulation of Bcl-2 protein expression.A novel role of autophagy in various cancers has been proposed.Interestingly,evidence that supports both a positive and negative role of autophagy in the pathogenesis of cancer has been reported.As a tumor suppression mechanism,autophagy maintains genome stability,induces senescence and possibly autophagic cell death.On the other hand,autophagy participates in tumor growth and maintenance by supplying metabolic substrate,limiting oxidative stress,and maintaining cancer stem cell population.It has been proposed that the differential roles of autophagy in cancer are disease type and stage specific.In addition,substrate selectivity might be involved in carrying out the specific effect of autophagy in cancer,and represents one of the potential directions for future studies.

  14. Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation

    Institute of Scientific and Technical Information of China (English)

    Guoliang Qing; Pengrong Yan; Zhaoxia Qu; Hudan Liu; Gutian Xiao

    2007-01-01

    NF-κB-inducing kinase (NIK) is required for NF-κB activation based on the processing of NF-κB2 p100. Here we report a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein (Hsp90) and autophagy.Functional inhibition of lisp90 by the anti-tumor agent geldanamycin (GA) efficiently disrupts its interaction with NIK,resulting in NIK degradation and subsequent blockage of p100 processing. Surprisingly, GA-induced NIK degradation is mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specifically involved in the folding/stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription and translation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the α subunit of IκB kinase to p100, a key step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradation and identify a novel function of autophagy in NF-κB regulation. These findings also suggest a new therapeutic strategy for diseases associated with p100 processing.

  15. Crystal Structure of Oxidative Stress Sensor Keap1 in Complex with Selective Autophagy Substrate p62

    Science.gov (United States)

    Kurokawa, Hirofumi

    Keap1, an adaptor protein of cullin-RING ubiquitin ligase complex, represses cytoprotective transcription factor Nrf2 in an oxidative stress-dependent manner. The accumulation of selective autophagy substrate p62 also activates Nrf2 target genes, but the detailed mechanism has not been elucidated. Crystal structure of Keap1-p62 complex revealed the structural basis for the Nrf2 activation in which Keap1 is inactivated by p62. The accumulation of p62 is observed in hepatocellular carcinoma. The activation of Nrf2 target genes, including detoxifying enzymes and efflux transporters, by p62 may protect the cancer cells from anti-cancer drugs.

  16. Targeting Autophagy Addiction in Cancer

    OpenAIRE

    Mancias, Joseph Douglas; Kimmelman, Alec C

    2011-01-01

    Autophagy inhibition is a novel cancer therapeutic strategy in the early stages of clinical trial testing. The initial rationale for using autophagy inhibition was generated by research revealing that autophagy is upregulated in response to external stresses, including chemotherapy and radiotherapy. Combining autophagy inhibition with agents that induce autophagy as a pro-survival response may therefore increase their therapeutic efficacy. Recent research has shown that some cancer cells, ...

  17. MicroRNA regulation of Autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lund, Anders H

    2012-01-01

    progress has recently contributed to our understanding of the molecular mechanisms of the autophagy machinery, yet several gaps remain in our knowledge of this process. The discovery of microRNAs (miRNAs) established a new paradigm of post-transcriptional gene regulation and during the past decade these...

  18. Autophagy contributes to resistance of tumor cells to ionizing radiation

    International Nuclear Information System (INIS)

    Background and purpose: Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. Materials and methods: Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. Results: LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. Conclusion: Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.

  19. Autophagy and Liver Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Raffaele Cursio

    2015-01-01

    Full Text Available Liver ischemia-reperfusion (I-R injury occurs during liver resection, liver transplantation, and hemorrhagic shock. The main mode of liver cell death after warm and/or cold liver I-R is necrosis, but other modes of cell death, as apoptosis and autophagy, are also involved. Autophagy is an intracellular self-digesting pathway responsible for removal of long-lived proteins, damaged organelles, and malformed proteins during biosynthesis by lysosomes. Autophagy is found in normal and diseased liver. Although depending on the type of ischemia, warm and/or cold, the dynamic process of liver I-R results mainly in adenosine triphosphate depletion and in production of reactive oxygen species (ROS, leads to both, a local ischemic insult and an acute inflammatory-mediated reperfusion injury, and results finally in cell death. This process can induce liver dysfunction and can increase patient morbidity and mortality after liver surgery and hemorrhagic shock. Whether autophagy protects from or promotes liver injury following warm and/or cold I-R remains to be elucidated. The present review aims to summarize the current knowledge in liver I-R injury focusing on both the beneficial and the detrimental effects of liver autophagy following warm and/or cold liver I-R.

  20. Cellular and Molecular Connections between Autophagy and Inflammation

    Directory of Open Access Journals (Sweden)

    Pierre Lapaquette

    2015-01-01

    Full Text Available Autophagy is an intracellular catabolic pathway essential for the recycling of proteins and larger substrates such as aggregates, apoptotic corpses, or long-lived and superfluous organelles whose accumulation could be toxic for cells. Because of its unique feature to engulf part of cytoplasm in double-membrane cup-shaped structures, which further fuses with lysosomes, autophagy is also involved in the elimination of host cell invaders and takes an active part of the innate and adaptive immune response. Its pivotal role in maintenance of the inflammatory balance makes dysfunctions of the autophagy process having important pathological consequences. Indeed, defects in autophagy are associated with a wide range of human diseases including metabolic disorders (diabetes and obesity, inflammatory bowel disease (IBD, and cancer. In this review, we will focus on interrelations that exist between inflammation and autophagy. We will discuss in particular how mediators of inflammation can regulate autophagy activity and, conversely, how autophagy shapes the inflammatory response. Impact of genetic polymorphisms in autophagy-related gene on inflammatory bowel disease will be also discussed.

  1. Autophagy and Autoimmunity CrossTalks

    Directory of Open Access Journals (Sweden)

    Abhisek eBhattacharya

    2013-04-01

    Full Text Available Autophagy, initially viewed as a conserved bulk-degradation mechanism, has emerged as a central player in a multitude of immune functions. Autophagy is important in host defense against intracellular and extracellular pathogens, metabolic syndromes, immune cell homeostasis, antigen processing and presentation and maintenance of tolerance. The observation that the above processes are implicated in triggering or exacerbating autoimmunity raises the possibility that the autophagy pathway is involved in mediating autoimmune processes, either directly or as a consequence of innate or adaptive functions mediated by the pathway. Genome-wide association studies have shown association between single nucleotide polymorphisms (SNPs in autophagy related gene 5 (Atg5, and Atg16l1 with susceptibility to systemic lupus erythematous (SLE and Crohn’s disease, respectively. Enhanced expression of Atg5 was also reported in blood of mice with experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis (MS, and in T cells isolated from blood or brain tissues from patients with active relapse of MS. This review explores the roles of autophagy pathway in the innate and adaptive immune systems on regulating or mediating the onset, progression or exacerbation of autoimmune processes.

  2. Autophagy in protists.

    Science.gov (United States)

    Duszenko, Michael; Ginger, Michael L; Brennand, Ana; Gualdrón-López, Melisa; Colombo, María Isabel; Coombs, Graham H; Coppens, Isabelle; Jayabalasingham, Bamini; Langsley, Gordon; de Castro, Solange Lisboa; Menna-Barreto, Rubem; Mottram, Jeremy C; Navarro, Miguel; Rigden, Daniel J; Romano, Patricia S; Stoka, Veronika; Turk, Boris; Michels, Paul A M

    2011-02-01

    Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target. PMID:20962583

  3. Autophagy: Friend or Foe in Breast Cancer Development, Progression, and Treatment

    International Nuclear Information System (INIS)

    Autophagy is a catabolic process responsible for the degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. Thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. On the other hand, this process also functions as a protective cell-survival mechanism against environmental stress causing resistance to antineoplastic therapies. Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings

  4. IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans.

    Science.gov (United States)

    de Luca, Antonella; Smeekens, Sanne P; Casagrande, Andrea; Iannitti, Rossana; Conway, Kara L; Gresnigt, Mark S; Begun, Jakob; Plantinga, Theo S; Joosten, Leo A B; van der Meer, Jos W M; Chamilos, Georgios; Netea, Mihai G; Xavier, Ramnik J; Dinarello, Charles A; Romani, Luigina; van de Veerdonk, Frank L

    2014-03-01

    Patients with chronic granulomatous disease (CGD) have a mutated NADPH complex resulting in defective production of reactive oxygen species; these patients can develop severe colitis and are highly susceptible to invasive fungal infection. In NADPH oxidase-deficient mice, autophagy is defective but inflammasome activation is present despite lack of reactive oxygen species production. However, whether these processes are mutually regulated in CGD and whether defective autophagy is clinically relevant in patients with CGD is unknown. Here, we demonstrate that macrophages from CGD mice and blood monocytes from CGD patients display minimal recruitment of microtubule-associated protein 1 light chain 3 (LC3) to phagosomes. This defect in autophagy results in increased IL-1β release. Blocking IL-1 with the receptor antagonist (anakinra) decreases neutrophil recruitment and T helper 17 responses and protects CGD mice from colitis and also from invasive aspergillosis. In addition to decreased inflammasome activation, anakinra restored autophagy in CGD mice in vivo, with increased Aspergillus-induced LC3 recruitment and increased expression of autophagy genes. Anakinra also increased Aspergillus-induced LC3 recruitment from 23% to 51% (P CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1-dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade. PMID:24550444

  5. Environmental Correlation Analysis for Genes Associated with Protection against Malaria.

    Science.gov (United States)

    Mackinnon, Margaret J; Ndila, Carolyne; Uyoga, Sophie; Macharia, Alex; Snow, Robert W; Band, Gavin; Rautanen, Anna; Rockett, Kirk A; Kwiatkowski, Dominic P; Williams, Thomas N

    2016-05-01

    Genome-wide searches for loci involved in human resistance to malaria are currently being conducted on a large scale in Africa using case-control studies. Here, we explore the utility of an alternative approach-"environmental correlation analysis, ECA," which tests for clines in allele frequencies across a gradient of an environmental selection pressure-to identify genes that have historically protected against death from malaria. We collected genotype data from 12,425 newborns on 57 candidate malaria resistance loci and 9,756 single nucleotide polymorphisms (SNPs) selected at random from across the genome, and examined their allele frequencies for geographic correlations with long-term malaria prevalence data based on 84,042 individuals living under different historical selection pressures from malaria in coastal Kenya. None of the 57 candidate SNPs showed significant (P ECA in the discovery of novel genes and pathways affecting infectious disease. PMID:26744416

  6. Lithium and autophagy.

    Science.gov (United States)

    Motoi, Yumiko; Shimada, Kohei; Ishiguro, Koichi; Hattori, Nobutaka

    2014-06-18

    Lithium, a drug used to treat bipolar disorders, has a variety of neuroprotective mechanisms, including autophagy regulation, in various neuropsychiatric conditions. In neurodegenerative diseases, lithium enhances degradation of aggregate-prone proteins, including mutated huntingtin, phosphorylated tau, and α-synuclein, and causes damaged mitochondria to degrade, while in a mouse model of cerebral ischemia and Alzheimer's disease autophagy downregulation by lithium is observed. The signaling pathway of lithium as an autophagy enhancer might be associated with the mammalian target of rapamycin (mTOR)-independent pathway, which is involved in myo-inositol-1,4,5-trisphosphate (IP3) in Huntington's disease and Parkinson's disease. However, the mTOR-dependent pathway might be involved in inhibiting glycogen synthase kinase-3β (GSK3β) in other diseases. Lithium's autophagy-enhancing property may contribute to the therapeutic benefit of patients with neuropsychiatric disorders. PMID:24738557

  7. Hypoxic Preconditioning Alleviates Ethanol Neurotoxicity: the Involvement of Autophagy

    OpenAIRE

    Wang, Haiping; Bower, Kimberly A.; Frank, Jacqueline A.; Xu, Mei; Luo, Jia

    2013-01-01

    Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. A sublethal preconditioning has been proposed as a neuroprotective strategy against several central nervous system (CNS) neurodegenerative diseases. We have recently demonstrated that autophagy is a protective response to alleviate ethanol toxicity. A modest hypoxic preconditioning (1% oxygen) did not cause neurotoxicity but induced autophagy (Tzeng et al., 2010). We the...

  8. Targeting autophagy in cancer management – strategies and developments

    Directory of Open Access Journals (Sweden)

    Ozpolat B

    2015-09-01

    Full Text Available Bulent Ozpolat,1 Doris M Benbrook2 1Department of Experimental Therapeutics, The University of Texas – Houston, MD Anderson Cancer Center, Houston, TX, 2Department of Obstetrics and Gynecology, University of Oklahoma HSC, Oklahoma City, OK, USA Abstract: Autophagy is a highly regulated catabolic process involving lysosomal degradation of intracellular components, damaged organelles, misfolded proteins, and toxic aggregates, reducing oxidative stress and protecting cells from damage. The process is also induced in response to various conditions, including nutrient deprivation, metabolic stress, hypoxia, anticancer therapeutics, and radiation therapy to adapt cellular conditions for survival. Autophagy can function as a tumor suppressor mechanism in normal cells and dysregulation of this process (ie, monoallelic Beclin-1 deletion may lead to malignant transformation and carcinogenesis. In tumors, autophagy is thought to promote tumor growth and progression by helping cells to adapt and survive in metabolically-challenged and harsh tumor microenvironments (ie, hypoxia and acidity. Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemo and radiation therapy. Currently, more than 30 clinical trials are investigating the effects of autophagy inhibition in combination with cytotoxic chemotherapies and targeted agents in various cancers. In this review, we will discuss the role, molecular mechanism, and regulation of autophagy, while targeting this process as a novel therapeutic modality, in various cancers. Keywords: autophagy inhibition, chemotherapy, tumor microenvironment

  9. The Regulation of Autophagy by Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Rong Zhang

    2014-01-01

    Full Text Available Influenza A virus is a dreadful pathogen of animals and humans, causing widespread infection and severe morbidity and mortality. It is essential to characterize the influenza A virus-host interaction and develop efficient counter measures against the viral infection. Autophagy is known as a catabolic process for the recycling of the cytoplasmic macromolecules. Recently, it has been shown that autophagy is a critical mechanism underlying the interaction between influenza A virus and its host. Autophagy can be induced by the infection with influenza A virus, which is considered as a necessary process for the viral proliferation, including the accumulation of viral elements during the replication of influenza A virus. On the other hand, influenza A virus can inhibit the autophagic formation via interaction with the autophagy-related genes (Atg and signaling pathways. In addition, autophagy is involved in the influenza virus-regulated cell deaths, leading to significant changes in host apoptosis. Interestingly, the high pathogenic strains of influenza A virus, such as H5N1, stimulate autophagic cell death and appear to interplay with the autophagy in distinct ways as compared with low pathogenic strains. This review discusses the regulation of autophagy, an influenza A virus driven process.

  10. Piperlongumine induces autophagy by targeting p38 signaling.

    Science.gov (United States)

    Wang, Y; Wang, J-W; Xiao, X; Shan, Y; Xue, B; Jiang, G; He, Q; Chen, J; Xu, H-G; Zhao, R-X; Werle, K D; Cui, R; Liang, J; Li, Y-L; Xu, Z-X

    2013-01-01

    Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death. PMID:24091667

  11. Transcriptional regulation of mammalian autophagy at a glance.

    Science.gov (United States)

    Füllgrabe, Jens; Ghislat, Ghita; Cho, Dong-Hyung; Rubinsztein, David C

    2016-08-15

    Macroautophagy, hereafter referred to as autophagy, is a catabolic process that results in the lysosomal degradation of cytoplasmic contents ranging from abnormal proteins to damaged cell organelles. It is activated  under diverse conditions, including nutrient deprivation and hypoxia. During autophagy, members of the core autophagy-related (ATG) family of proteins mediate membrane rearrangements, which lead to the engulfment and degradation of cytoplasmic cargo. Recently, the nuclear regulation of autophagy, especially by transcription factors and histone modifiers, has gained increased attention. These factors are not only involved in rapid responses to autophagic stimuli, but also regulate the long-term outcome of autophagy. Now there are more than 20 transcription factors that have been shown to be linked to the autophagic process. However, their interplay and timing appear enigmatic as several have been individually shown to act as major regulators of autophagy. This Cell Science at a Glance article and the accompanying poster highlights the main cellular regulators of transcription involved in mammalian autophagy and their target genes. PMID:27528206

  12. Baicalein pretreatment reduces liver ischemia/reperfusion injury via induction of autophagy in rats.

    Science.gov (United States)

    Liu, Anding; Huang, Liang; Guo, Enshuang; Li, Renlong; Yang, Jiankun; Li, Anyi; Yang, Yan; Liu, Shenpei; Hu, Jifa; Jiang, Xiaojing; Dirsch, Olaf; Dahmen, Uta; Sun, Jian

    2016-01-01

    We previously demonstrated that baicalein could protect against liver ischemia/reperfusion (I/R) injury in mice. The exact mechanism of baicalein remains poorly understood. Autophagy plays an important role in protecting against I/R injury. This study was designed to determine whether baicalein could protect against liver I/R injury via induction of autophagy in rats. Baicalein was intraperitoneally injected 1 h before warm ischemia. Pretreatment with baicalein prior to I/R insult significantly blunted I/R-induced elevations of serum aminotransferase levels and significantly improved the histological status of livers. Electron microscopy and expression of the autophagic marker LC3B-II suggested induction of autophagy after baicalein treatment. Moreover, inhibition of the baicalein-induced autophagy using 3-methyladenine (3-MA) worsened liver injury. Furthermore, baicalein treatment increased heme oxygenase (HO)-1 expression, and pharmacological inhibition of HO-1 with tin protoporphyrin IX (SnPP) abolished the baicalein-mediated autophagy and the hepatocellular protection. In primary rat hepatocytes, baicalein-induced autophagy also protected hepatocytes from hypoxia/reoxygenation injury in vitro and the beneficial effect was abrogated by 3-MA or Atg7 siRNA, respectively. Suppression of HO-1 activity by SnPP or HO-1 siRNA prevented the baicalein-mediated autophagy and resulted in increased hepatocellular injury. Collectively, these results suggest that baicalein prevents hepatocellular injury via induction of HO-1-mediated autophagy. PMID:27150843

  13. Autophagy: a new target for nonalcoholic fatty liver disease therapy

    Directory of Open Access Journals (Sweden)

    Mao YQ

    2016-03-01

    Full Text Available Yuqing Mao,1 Fujun Yu,1 Jianbo Wang,2 Chuanyong Guo,3 Xiaoming Fan1 1Department of Gastroenterology and Hepatology, Jinshan Hospital of Fudan University, Shanghai, 2Department of Gastroenterology and Hepatology, The Central Hospital of Lishui City, Wenzhou Medical University, Zhejiang, 3Department of Gastroenterology and Hepatology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, People's Republic of China Abstract: Nonalcoholic fatty liver disease (NAFLD has gained importance in recent decades due to drastic changes in diet, especially in Western countries. NAFLD occurs as a spectrum from simple hepatic steatosis, steatohepatitis to cirrhosis, and even hepatocellular carcinoma. Although the molecular mechanisms underlying the development of NAFLD have been intensively investigated, many issues remain to be resolved. Autophagy is a cell survival mechanism for disposing of excess or defective organelles, and has become a hot spot for research. Recent studies have revealed that autophagy is linked to the development of NAFLD and regulation of autophagy has therapeutic potential. Autophagy reduces intracellular lipid droplets by enclosing them and fusing with lysosomes for degradation. Furthermore, autophagy is involved in attenuating inflammation and liver injury. However, autophagy is regarded as a double-edged sword, as it may also affect adipogenesis and adipocyte differentiation. Moreover, it is unclear as to whether autophagy protects the body from injury or causes diseases and even death, and the association between autophagy and NAFLD remains controversial. This review is intended to discuss, comment, and outline the progress made in this field and establish the possible molecular mechanism involved. Keywords: nonalcoholic fatty liver disease, autophagy, steatosis, steatohepatitis, fibrosis, carcinogenesis

  14. The interplays between autophagy and apoptosis induced by enterovirus 71.

    Directory of Open Access Journals (Sweden)

    Xueyan Xi

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I to LC3-II and degradation of sequestosome 1 (SQSTM1/P62. Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles. CONCLUSIONS/SIGNIFICANCE: In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.

  15. Avermectin induced autophagy in pigeon spleen tissues.

    Science.gov (United States)

    Liu, Ci; Zhao, Yanbing; Chen, Lijie; Zhang, Ziwei; Li, Ming; Li, Shu

    2015-12-01

    The level of autophagy is considered as an indicator for monitoring the toxic impact of pesticide exposure. Avermectin (AVM), a widely used insecticide, has immunotoxic effects on the pigeon spleen. The aim of this study was to investigate the status of autophagy and the expression levels of microtubule-associated protein1 light chain 3 (LC3), beclin-1, dynein, autophagy associated gene (Atg) 4B, Atg5, target of rapamycin complex 1 (TORC1) and target of rapamycin complex 2 (TORC2) in AVM-treated pigeon spleens. Eighty two-month-old pigeons were randomly divided into four groups: a control group, a low-dose group, a medium-dose group and a high-dose group, which were fed a basal diet spiked with 0, 20, 40 and 60 mg AVM/kg diet, respectively. Microscopic cellular morphology revealed a significant increase in autophagic structures in the AVM-treated groups. The expression of LC3, beclin-1, dynein, Atg4B and Atg5 increased, while mRNA levels of TORC1 and TORC2 were decreased in the AVM-treated groups relative to the control groups at 30, 60 and 90 days in the pigeon spleen. These results indicated that AVM exposure could up-regulate the level of autophagy in a dose-time-dependent manner in the pigeon spleen. PMID:26541719

  16. Receptor Proteins in Selective Autophagy

    Directory of Open Access Journals (Sweden)

    Christian Behrends

    2012-01-01

    Full Text Available Autophagy has long been thought to be an essential but unselective bulk degradation pathway. However, increasing evidence suggests selective autophagosomal turnover of a broad range of substrates. Bifunctional autophagy receptors play a key role in selective autophagy by tethering cargo to the site of autophagosomal engulfment. While the identity of molecular components involved in selective autophagy has been revealed at least to some extent, we are only beginning to understand how selectivity is achieved in this process. Here, we summarize the mechanistic and structural basis of receptor-mediated selective autophagy.

  17. Dysregulation of the Autophagy-Endolysosomal System in Amyotrophic Lateral Sclerosis and Related Motor Neuron Diseases

    Directory of Open Access Journals (Sweden)

    Asako Otomo

    2012-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a heterogeneous group of incurable motor neuron diseases (MNDs characterized by a selective loss of upper and lower motor neurons in the brain and spinal cord. Most cases of ALS are sporadic, while approximately 5–10% cases are familial. More than 16 causative genes for ALS/MNDs have been identified and their underlying pathogenesis, including oxidative stress, endoplasmic reticulum stress, excitotoxicity, mitochondrial dysfunction, neural inflammation, protein misfolding and accumulation, dysfunctional intracellular trafficking, abnormal RNA processing, and noncell-autonomous damage, has begun to emerge. It is currently believed that a complex interplay of multiple toxicity pathways is implicated in disease onset and progression. Among such mechanisms, ones that are associated with disturbances of protein homeostasis, the ubiquitin-proteasome system and autophagy, have recently been highlighted. Although it remains to be determined whether disease-associated protein aggregates have a toxic or protective role in the pathogenesis, the formation of them results from the imbalance between generation and degradation of misfolded proteins within neuronal cells. In this paper, we focus on the autophagy-lysosomal and endocytic degradation systems and implication of their dysfunction to the pathogenesis of ALS/MNDs. The autophagy-endolysosomal pathway could be a major target for the development of therapeutic agents for ALS/MNDs.

  18. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Orfali, Nina [Cork Cancer Research Center, University College Cork, Cork (Ireland); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); McKenna, Sharon L. [Cork Cancer Research Center, University College Cork, Cork (Ireland); Cahill, Mary R. [Department of Hematology, Cork University Hospital, Cork (Ireland); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); Mongan, Nigel P., E-mail: nigel.mongan@nottingham.ac.uk [Faculty of Medicine and Health Science, School of Veterinary Medicine and Science, University of Nottingham, LE12 5RD (United Kingdom); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States)

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.

  19. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    International Nuclear Information System (INIS)

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects

  20. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

    DEFF Research Database (Denmark)

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V; Eisenberg, Tobias; Megalou, Evgenia; Schroeder, Sabrina; Cabrera, Sandra; Bénit, Paule; Rustin, Pierre; Criollo, Alfredo; Kepp, Oliver; Galluzzi, Lorenzo; Shen, Shensi; Malik, Shoaib Ahmad; Maiuri, Maria Chiara; Horio, Yoshiyuki; López-Otín, Carlos; Andersen, Jens S.; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2011-01-01

    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy...... independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation...... and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions...

  1. Targeting autophagy as a potential therapeutic approach for immune thrombocytopenia therapy.

    Science.gov (United States)

    Shan, Ning-Ning; Dong, Li-Li; Zhang, Xiao-Mei; Liu, Xin; Li, Ying

    2016-04-01

    Autophagy involves the sequestration and lysosomal degradation of various cytoplasmic structures, including damaged organelles and invading microorganisms. Autophagy is not only an essential cell-intrinsic mechanism for protecting against internal and external stress conditions but is also key in the cellular response against microbes, in antigen processing for major histocompatibility complex (MHC) presentation, and in lymphocyte development, survival, and proliferation. In recent years, perturbations in autophagy have been implicated in a number of diseases, including autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Immune thrombocytopenia (ITP) is a multifactorial disease characterized by autoimmune responses to self-platelet membrane proteins. Recently, our unpublished original data demonstrated aberrant expression of molecules in the autophagy pathway in ITP patients compared with controls, and we found a close correlation between the pathogenesis of ITP and the autophagy pathway. The potential of targeting the autophagy pathway in ITP as a novel therapeutic approach has been discussed. PMID:26830007

  2. The role of autophagy in cell survival from heavy ion irradiation in the plateau region

    International Nuclear Information System (INIS)

    To study cytotoxic effect of heavy ion irradiation in the plateau region, and investigate whether autophagy induced by heavy ion irradiation is cytoprotective, HeLa cells were irradiated with 350 MeV/u carbon ions beams, and the clonogenic survival was analyzed. The results showed that cell survival decreased with increasing doses. It was also found that G2/M-phase cells increased, and the autophagy-related activity was significantly higher than the control. When autophagy was blocked by 3-methyladenine in carbon-ion irradiated cells, G2/M phase arrest and the percentage of apoptosis cells were further elevated, and cell survival decreased significantly, indicating the induction of cytoprotective autophagy by carbon-ion irradiation. Our results demonstrated that autophagy induced by carbon ion irradiation provided a self-protective mechanism in HeLa cells, short-time inhibition of autophagy before carbon-ion irradiation could enhance radiation cytotoxicity in HeLa cells. (authors)

  3. SIRT6 suppresses isoproterenol-induced cardiac hypertrophy through activation of autophagy.

    Science.gov (United States)

    Lu, Jing; Sun, Duanping; Liu, Zhiping; Li, Min; Hong, Huiqi; Liu, Cui; Gao, Si; Li, Hong; Cai, Yi; Chen, Shaorui; Li, Zhuoming; Ye, Jiantao; Liu, Peiqing

    2016-06-01

    Reduction in autophagy has been reported to contribute to the pathogenesis of cardiac hypertrophy. However, the molecular pathways leading to impaired autophagy at the presence of hypertrophic stimuli remain to be elucidated. The present study aimed to investigate the role of sirtuin 6 (SIRT6), a sirtuin family member, in regulating cardiomyocyte autophagy, and its implication in prevention of cardiac hypertrophy. Primary neonatal rat cardiomyocytes (NRCMs) or Sprague-Dawley (SD) rats were submitted to isoproterenol (ISO) treatment, and then the hypertrophic responses and changes in autophagy activity were measured. The influence of SIRT6 on autophagy was observed in cultured NRCMs with gain- and loss-of-function approaches to regulate SIRT6 expression, and further confirmed in vivo by intramyocardial delivery of an adenovirus vector encoding SIRT6 cDNA. In addition, the involvement of SIRT6-mediated autophagy in attenuation of cardiomyocyte hypertrophy induced by ISO was determined basing on genetic or pharmaceutical disruption of autophagy, and the underlying mechanism was preliminarily explored. ISO-caused cardiac hypertrophy accompanying with a significant decrease in autophagy activity. SIRT6 overexpression enhanced autophagy in NRCMs and in rat hearts, whereas knockdown of SIRT6 by RNA interference led to suppression of cardiomyocyte autophagy. Furthermore, the protective effect of SIRT6 against ISO-stimulated hypertrophy was associated with induction of autophagy. SIRT6 promoted nuclear retention of forkhead box O3 transcription factor possibly via attenuating Akt signaling, which was responsible for autophagy activation. Our findings revealed that SIRT6 positively regulates autophagy in cardiomyocytes, which may help to ameliorate ISO-induced cardiac hypertrophy. PMID:27016702

  4. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    International Nuclear Information System (INIS)

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/β-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome–lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  5. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Yang, Hanchun, E-mail: yanghanchun1@cau.edu.cn [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Hu, Hongbo, E-mail: hongbo@cau.edu.cn [College of Food Science and Nutritional Engineering, China Agricultural University, Beijing (China)

    2012-08-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/{beta}-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome-lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  6. Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

    Directory of Open Access Journals (Sweden)

    Sung Won Lee

    Full Text Available Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence

  7. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  8. Dopamine Oxidation and Autophagy

    Directory of Open Access Journals (Sweden)

    Patricia Muñoz

    2012-01-01

    Full Text Available The molecular mechanisms involved in the neurodegenerative process of Parkinson's disease remain unclear. Currently, there is a general agreement that mitochondrial dysfunction, α-synuclein aggregation, oxidative stress, neuroinflammation, and impaired protein degradation are involved in the neurodegeneration of dopaminergic neurons containing neuromelanin in Parkinson's disease. Aminochrome has been proposed to play an essential role in the degeneration of dopaminergic neurons containing neuromelanin by inducing mitochondrial dysfunction, oxidative stress, the formation of neurotoxic α-synuclein protofibrils, and impaired protein degradation. Here, we discuss the relationship between the oxidation of dopamine to aminochrome, the precursor of neuromelanin, autophagy dysfunction in dopaminergic neurons containing neuromelanin, and the role of dopamine oxidation to aminochrome in autophagy dysfunction in dopaminergic neurons. Aminochrome induces the following: (i the formation of α-synuclein protofibrils that inactivate chaperone-mediated autophagy; (ii the formation of adducts with α- and β-tubulin, which induce the aggregation of the microtubules required for the fusion of autophagy vacuoles and lysosomes.

  9. Streptococcus pneumoniae induces pyroptosis through the regulation of autophagy in murine microglia.

    Science.gov (United States)

    Kim, Ji-Yun; Paton, James C; Briles, David E; Rhee, Dong-Kwon; Pyo, Suhkneung

    2015-12-29

    Streptococcus pneumoniae is responsible for significant mortality and morbidity worldwide and causes invasive pneumococcal diseases including pneumococcal meningitis. Pyroptosis is caspase-1-dependent inflammatory cell death and is known to be induced by various microbial infections. In the present study, we investigated the molecular mechanisms that regulate pyroptosis induced by S. pneumoniae in microglia. Our results revealed that S. pneumoniae induced pyroptosis through caspase-1 activation and IL-1β production. We also found that the activation of caspase-1 and the maturation of IL-1β and IL-18 in the S. pneumoniae-triggered pyroptotic cell death process were mediated by NLRP3 inflammasome. In addition, pneumococcal infection increased the expression of autophagy-related genes and induced autophagosome formation. We also showed that the inhibition of autophagy promoted pneumococcus-induced pyroptosis. Furthermore, ROS was generated by pneumococcal infection and inhibited caspase-1 activation within 4 h of infection. However, in the late phase of infection, IL-1β secretion and caspase-1-dependent cell death were induced by ROS. These results suggest that autophagy induction transiently delay pyroptosis induced by S. pneumoniae in microglia. Our study also revealed that the activation of caspase-1 and the production of IL-1β were induced by pneumolysin and that pneumolysin triggered pyroptosis in microglial cells. Similar to the in vitro results, S. pneumoniae induced caspase-1 activation and caspase-1-dependent cytokine maturation in the mouse meningitis model. Thus, the present data demonstrate that S. pneumoniae induces pyroptosis in murine microglia and that NLRP3 inflammasome is critical for caspase-1 activation during the process. Furthermore, the induction of autophagy could transiently protect microglia from pyroptosis. PMID:26683708

  10. Targeting autophagy in cancer management – strategies and developments

    International Nuclear Information System (INIS)

    Autophagy is a highly regulated catabolic process involving lysosomal degradation of intracellular components, damaged organelles, misfolded proteins, and toxic aggregates, reducing oxidative stress and protecting cells from damage. The process is also induced in response to various conditions, including nutrient deprivation, metabolic stress, hypoxia, anticancer therapeutics, and radiation therapy to adapt cellular conditions for survival. Autophagy can function as a tumor suppressor mechanism in normal cells and dysregulation of this process (ie, monoallelic Beclin-1 deletion) may lead to malignant transformation and carcinogenesis. In tumors, autophagy is thought to promote tumor growth and progression by helping cells to adapt and survive in metabolically-challenged and harsh tumor microenvironments (ie, hypoxia and acidity). Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemo and radiation therapy. Currently, more than 30 clinical trials are investigating the effects of autophagy inhibition in combination with cytotoxic chemotherapies and targeted agents in various cancers. In this review, we will discuss the role, molecular mechanism, and regulation of autophagy, while targeting this process as a novel therapeutic modality, in various cancers

  11. Role of autophagy in prion protein-induced neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Hao Yao; Deming Zhao; Sher Hayat Khan; Lifeng Yang

    2013-01-01

    Prion diseases,characterized by spongiform degeneration and the accumulation of misfolded and aggregated PrPSc in the central nervous system,are one of fatal neurodegenerative and infectious disorders of humans and animals.In earlier studies,autophagy vacuoles in neurons were frequently observed in neurodegenerative diseases such as Alzheimer's,Parkinson's,and Huntington's diseases as well as prion diseases.Autophagy is a highly conserved homeostatic process by which several cytoplasmic components (proteins or organelles) are sequestered in a doublemembrane-bound vesicle termed 'autophagosome' and degraded upon their fusion with lysosome.The pathway of intercellular self-digestion at basal physiological levels is indispensable for maintaining the healthy status of tissues and organs.In case of prion infection,increasing evidence indicates that autophagy has a crucial ability of eliminating pathological PrPSc accumulated within neurons.In contrast,autophagy dysfunction in affected neurons may contribute to the formation of spongiform changes.In this review,we summarized recent findings about the effect of mammalian autophagy in neurodegenerative disorders,particularly in prion diseases.We also summarized the therapeutic potential of some small molecules (such as lithium,rapamycin,Sirtuin 1 and resveratrol) targets to mitigate such diseases on brain function.Furthermore,we discussed the controversial role of autophagy,whether it mediates neuronal toxicity or serves a protective function in neurodegenerative disorders.

  12. Autophagy and ethanol-induced liver injury

    Institute of Scientific and Technical Information of China (English)

    Terrence M Donohue Jr

    2009-01-01

    The majority of ethanol metabolism occurs in the liver. Consequently, this organ sustains the greatest damage from ethanol abuse. Ethanol consumption disturbs the delicate balance of protein homeostasis in the liver, causing intracellular protein accumulation due to a disruption of hepatic protein catabolism.Evidence indicates that ethanol or its metabolism impairs trafficking events in the liver, including the process of macroautophagy, which is the engulfment and degradation of cytoplasmic constituents by the lysosomal system. Autophagy is an essential, ongoing cellular process that is highly regulated by nutrients,endocrine factors and signaling pathways. A great number of the genes and gene products that govern the autophagic response have been characterized and the major metabolic and signaling pathways that activate or suppress autophagy have been identified. This review describes the process of autophagy, its regulation and the possible mechanisms by which ethanol disrupts the process of autophagic degradation. The implications of autophagic suppression are discussed in relation to the pathogenesis of alcohol-induced liver injury.

  13. Autophagy in cardiovascular biology

    OpenAIRE

    Lavandero, Sergio; Chiong, Mario; Rothermel, Beverly A.; Hill, Joseph A.

    2015-01-01

    Cardiovascular disease is the leading cause of death worldwide. As such, there is great interest in identifying novel mechanisms that govern the cardiovascular response to disease-related stress. First described in failing hearts, autophagy within the cardiovascular system has been widely characterized in cardiomyocytes, cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, and macrophages. In all cases, a window of optimal autophagic activity appears to be critical to the mai...

  14. AUTOPHAGY IN LUNG CANCER

    OpenAIRE

    Jaboin, Jerry J.; Hwang, Misun; Lu, Bo

    2009-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. The relatively poor cure rate in lung cancer patients has been associated with a resistance to chemotherapy and radiation that is at least in part related to defects in cellular apoptotic machinery. Exploitation of another form of cell death, autophagy, has the capacity to improve the therapeutic gain of current therapies. In an effort to develop novel treatment strategies to enhance the therapeutic ratio for lung cancer, we...

  15. C1q/TNF-Related Protein 9 (CTRP9) attenuates hepatic steatosis via the autophagy-mediated inhibition of endoplasmic reticulum stress.

    Science.gov (United States)

    Jung, Tae Woo; Hong, Ho Cheol; Hwang, Hwan-Jin; Yoo, Hye Jin; Baik, Sei Hyun; Choi, Kyung Mook

    2015-12-01

    C1q/TNF-Related Protein (CTRP) 9, the closest paralog of adiponectin, has been reported to protect against diet-induced obesity and non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism has not been fully elucidated. We explored the protective effect of CTRP9 against hepatic steatosis and apoptosis, and identified the mechanisms through autophagy and endoplasmic reticulum (ER) stress using in vitro and in vivo experiments. Treating HepG2 cells with human recombinant CTRP9 significantly ameliorated palmitate- or tunicamycin-induced dysregulation of lipid metabolism, caspase 3 activity and chromatin condensation, which lead to reduction of hepatic triglyceride (TG) accumulation. CTRP9 treatment induced autophagy markers including LC3 conversion, P62 degradation, Beclin1 and ATG7 through AMPK phosphorylation in human primary hepatocytes. Furthermore, CTRP9 decreased palmitate- or tunicamycin-induced ER stress markers, such as eIF2α, CHOP and IRE-1, in HepG2 cells. Compound C, an AMPK inhibitor, and 3 methyladenine (3 MA), an autophagy inhibitor, canceled the effects of CTRP9 on ER stress, apoptosis and hepatic steatosis. In the livers of HFD-fed mice, adenovirus-mediated CTRP9 overexpression significantly induced AMPK phosphorylation and autophagy, whereas suppressed ER stress markers. In addition, both SREBP1-mediated lipogenic gene expression and apoptosis were significantly attenuated, which result in improvement in hepatic steatosis by overexpression of CTRP9. These results demonstrate that CTRP9 alleviates hepatic steatosis through relief of ER stress via the AMPK-mediated induction of autophagy. PMID:26419929

  16. Autophagy in dementias.

    Science.gov (United States)

    Kragh, Christine Lund; Ubhi, Kiren; Wyss-Coray, Tony; Wyss-Corey, Tony; Masliah, Eliezer

    2012-01-01

    Dementias are a varied group of disorders typically associated with memory loss, impaired judgment and/or language and by symptoms affecting other cognitive and social abilities to a degree that interferes with daily functioning. Alzheimer's disease (AD) is the most common cause of a progressive dementia, followed by dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), (VaD) and HIV-associated neurocognitive disorders (HAND). The pathogenesis of this group of disorders has been linked to the abnormal accumulation of proteins in the brains of affected individuals, which in turn has been related to deficits in protein clearance. Autophagy is a key cellular protein clearance pathway with proteolytic cleavage and degradation via the ubiquitin-proteasome pathway representing another important clearance mechanism. Alterations in the levels of autophagy and the proteins associated with the autophagocytic pathway have been reported in various types of dementias. This review will examine recent literature across these disorders and highlight a common theme of altered autophagy across the spectrum of the dementias. PMID:22150925

  17. Dexamethasone-induced autophagy mediates muscle atrophy through mitochondrial clearance

    Science.gov (United States)

    Troncoso, Rodrigo; Paredes, Felipe; Parra, Valentina; Gatica, Damián; Vásquez-Trincado, César; Quiroga, Clara; Bravo-Sagua, Roberto; López-Crisosto, Camila; Rodriguez, Andrea E; Oyarzún, Alejandra P; Kroemer, Guido; Lavandero, Sergio

    2014-01-01

    Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program. PMID:24897381

  18. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  19. Novel enabling technologies of gene isolation and plant transformation for improved crop protection

    Energy Technology Data Exchange (ETDEWEB)

    Torok, Tamas

    2013-02-04

    Meeting the needs of agricultural producers requires the continued development of improved transgenic crop protection products. The completed project focused on developing novel enabling technologies of gene discovery and plant transformation to facilitate the generation of such products.

  20. Structure biology of selective autophagy receptors

    Science.gov (United States)

    Kim, Byeong-Won; Kwon, Do Hoon; Song, Hyun Kyu

    2016-01-01

    Autophagy is a process tightly regulated by various autophagy-related proteins. It is generally classified into non-selective and selective autophagy. Whereas non-selective autophagy is triggered when the cell is under starvation, selective autophagy is involved in eliminating dysfunctional organelles, misfolded and/or ubiquitylated proteins, and intracellular pathogens. These components are recognized by autophagy receptors and delivered to phagophores. Several selective autophagy receptors have been identified and characterized. They usually have some common domains, such as motif, a specific cargo interacting (ubiquitin-dependent or ubiquitin-independent) domain. Recently, structural data of these autophagy receptors has been described, which provides an insight of their function in the selective autophagic process. In this review, we summarize the most up-to-date findings about the structure-function of autophagy receptors that regulates selective autophagy. [BMB Reports 2016; 49(2): 73-80] PMID:26698872

  1. Live and Let Die: Roles of Autophagy in Cadmium Nephrotoxicity

    Directory of Open Access Journals (Sweden)

    Frank Thévenod

    2015-04-01

    Full Text Available The transition metal ion cadmium (Cd2+ is a significant environmental contaminant. With a biological half-life of ~20 years, Cd2+ accumulates in the kidney cortex, where it particularly damages proximal tubule (PT cells and can result in renal fibrosis, failure, or cancer. Because death represents a powerful means by which cells avoid malignant transformation, it is crucial to clearly identify and understand the pathways that determine cell fate in chronic Cd2+ nephrotoxicity. When cells are subjected to stress, they make a decision to adapt and survive, or—depending on the magnitude and duration of stress—to die by several modes of death (programmed cell death, including autophagic cell death (ACD. Autophagy is part of a larger system of intracellular protein degradation and represents the channel by which organelles and long-lived proteins are delivered to the lysosome for degradation. Basal autophagy levels in all eukaryotic cells serve as a dynamic physiological recycling system, but they can also be induced by intra- or extracellular stress and pathological processes, such as endoplasmic reticulum (ER stress. In a context-dependent manner, autophagy can either be protective and hence contribute to survival, or promote death by non-apoptotic or apoptotic pathways. So far, the role of autophagy in Cd2+-induced nephrotoxicity has remained unsettled due to contradictory results. In this review, we critically survey the current literature on autophagy in Cd2+-induced nephrotoxicity in light of our own ongoing studies. Data obtained in kidney cells illustrate a dual and complex function of autophagy in a stimulus- and time-dependent manner that possibly reflects distinct outcomes in vitro and in vivo. A better understanding of the context-specific regulation of cell fate by autophagy may ultimately contribute to the development of preventive and novel therapeutic strategies for acute and chronic Cd2+ nephrotoxicity.

  2. Autophagy is required for stem cell mobilization by G-CSF

    DEFF Research Database (Denmark)

    Leveque-El Mouttie, Lucie; Vu, Therese; Lineburg, Katie E.;

    2015-01-01

    Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects...... the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF-induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy...... is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically...

  3. Crocin-Elicited Autophagy Rescues Myocardial Ischemia/Reperfusion Injury via Paradoxical Mechanisms.

    Science.gov (United States)

    Zeng, Chao; Li, Hu; Fan, Zhiwen; Zhong, Lei; Guo, Zhen; Guo, Yaping; Xi, Yusheng

    2016-01-01

    Crocin, the main effective component of saffron, exerts protective effects against ischemia/reperfusion injury during strokes. However, the effects of crocin in myocardial ischemia/reperfusion injury, and the mechanisms involved, remain unknown. Pretreated with crocin for 7 days, C57BL/6N mice were subjected to 30 min of myocardial ischemia followed by 12[Formula: see text]h of reperfusion (for cardiac function and infarct size, cell apoptosis and necrosis). Neonatal mouse cardiomyocytes were subjected to 2 h of hypoxia followed by 4 h of reoxygenation. NMCM's survival was assessed during hypoxia and reoxygenation in the presence or absence of the autophagy inhibitor 3-methyladenine or the inducer rapamycin. Western blotting was used to evaluate AMPK, Akt, and autophagy-related proteins. Autophagosome was observed using electron microscopy. In the in vivo experiment, crocin pretreatment significantly attenuated infarct size, myocardial apoptosis and necrosis, and improved left ventricular function following ischemia/reperfusion. In vitro data revealed that autophagy was induced during hypoxia, the levels of which were intensely elevated during reoxygenation. Crocin significantly promoted autophagy during ischemia, accompanied with the activation of AMPK. In contrast, crocin overtly inhibited autophagy during reperfusion, accompanied with Akt activation. Induction and inhibition of autophagy mitigated crocin induced protection against NMCMs injury during hypoxia and reoxygenation, respectively. Our data suggest that crocin demonstrated a myocardial protective effect via AMPK/mTOR and Akt/mTOR regulated autophagy against ischemia and reperfusion injury, respectively. PMID:27109157

  4. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    International Nuclear Information System (INIS)

    Highlights: ► We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. ► Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. ► Autophagy was required for normal terminal differentiation of preputial gland cells. ► Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  5. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Sukseree, Supawadee [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Rossiter, Heidemarie; Mildner, Michael [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Pammer, Johannes [Institute of Clinical Pathology, Medical University of Vienna, Vienna (Austria); Buchberger, Maria; Gruber, Florian [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Watanapokasin, Ramida [Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Tschachler, Erwin [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Eckhart, Leopold, E-mail: leopold.eckhart@meduniwien.ac.at [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  6. Activation of antibacterial autophagy by NADPH oxidases

    OpenAIRE

    Huang, Ju; Canadien, Veronica; Lam, Grace Y.; Steinberg, Benjamin E.; Mary C. Dinauer; Magalhaes, Marco A. O.; Glogauer, Michael; Grinstein, Sergio; Brumell, John H.

    2009-01-01

    Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR l...

  7. Autophagy and neurodegenerative disorders

    Institute of Scientific and Technical Information of China (English)

    Evangelia Kesidou; Roza Lagoudaki; Olga Touloumi; Kyriaki-Nefeli Poulatsidou; Constantina Simeonidou

    2013-01-01

    Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracel ular mechanism that removes damaged organelles and misfolded proteins in order to maintain cel homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.

  8. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang [East Hospital, Tongji University School of Medicine, Shanghai (China); Dong, Chuanming [East Hospital, Tongji University School of Medicine, Shanghai (China); Department of Anatomy and Neurobiology, The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong (China); Sun, Chenxi; Ma, Rongjie; Yang, Danjing [East Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Hongwen, E-mail: hongwen_zhu@hotmail.com [Tianjin Hospital, Tianjin Academy of Integrative Medicine, Tianjin (China); Xu, Jun, E-mail: xunymc2000@yahoo.com [East Hospital, Tongji University School of Medicine, Shanghai (China)

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  9. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    International Nuclear Information System (INIS)

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy

  10. You are what you eat: multifaceted functions of autophagy during C. elegans development.

    Science.gov (United States)

    Yang, Peiguo; Zhang, Hong

    2014-01-01

    Autophagy involves the sequestration of a portion of the cytosolic contents in an enclosed double-membrane autophagosomal structure and its subsequent delivery to lysosomes for degradation. Autophagy activity functions in multiple biological processes during Caenorhabditis elegans development. The basal level of autophagy in embryos removes aggregate-prone proteins, paternal mitochondria and spermatid-specific membranous organelles (MOs). Autophagy also contributes to the efficient removal of embryonic apoptotic cell corpses by promoting phagosome maturation. During larval development, autophagy modulates miRNA-mediated gene silencing by selectively degrading AIN-1, a component of miRNA-induced silencing complex, and thus participates in the specification of multiple cell fates controlled by miRNAs. During development of the hermaphrodite germline, autophagy acts coordinately with the core apoptotic machinery to execute genotoxic stress-induced germline cell death and also cell death when caspase activity is partially compromised. Autophagy is also involved in the utilization of lipid droplets in the aging process in adult animals. Studies in C. elegans provide valuable insights into the physiological functions of autophagy in the development of multicellular organisms. PMID:24296782

  11. Regulatory coordination between two major intracellular homeostatic systems: heat shock response and autophagy.

    Science.gov (United States)

    Dokladny, Karol; Zuhl, Micah Nathaniel; Mandell, Michael; Bhattacharya, Dhruva; Schneider, Suzanne; Deretic, Vojo; Moseley, Pope Lloyd

    2013-05-24

    The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell. PMID:23576438

  12. Autophagy precedes apoptosis during the remodeling of silkworm larval midgut.

    Science.gov (United States)

    Franzetti, Eleonora; Huang, Zhi-Jun; Shi, Yan-Xia; Xie, Kun; Deng, Xiao-Juan; Li, Jian-Ping; Li, Qing-Rong; Yang, Wan-Ying; Zeng, Wen-Nian; Casartelli, Morena; Deng, Hui-Min; Cappellozza, Silvia; Grimaldi, Annalisa; Xia, Qingyou; Feng, Qili; Cao, Yang; Tettamanti, Gianluca

    2012-03-01

    Although several features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two processes and the nature of their relationship are still cryptic. In this study, we perform a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut of Bombyx mori during larval-adult transformation, with the aim to analyze autophagy and apoptosis in cells that die under physiological conditions. We demonstrate that larval midgut degradation is due to the concerted action of the two mechanisms, which occur at different times and have different functions. Autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages, as demonstrated by specific autophagic markers. Our data show that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells. PMID:22127643

  13. Nucleobase protection strategy for gene cloning and expression

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Brock, N. L.; Dickschat, J. S.; Hocek, Michal

    2013-01-01

    Roč. 14, č. 7 (2013), s. 801-804. ISSN 1439-4227 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : DNA * polymerase chain reaction * protecting groups * protein production * restriction endonucleases Subject RIV: CE - Biochemistry Impact factor: 3.060, year: 2013

  14. Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

    Directory of Open Access Journals (Sweden)

    Hye Rin Lee

    Full Text Available Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ and the aggregated proteins that cause Alzheimer's disease (AD. We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5, and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM or resveratrol (20 μM prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition. Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion. After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein, cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

  15. Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment.

    Science.gov (United States)

    Rausch, Vanessa; Liu, Li; Apel, Anja; Rettig, Theresa; Gladkich, Jury; Labsch, Sabrina; Kallifatidis, Georgios; Kaczorowski, Adam; Groth, Ariane; Gross, Wolfgang; Gebhard, Martha M; Schemmer, Peter; Werner, Jens; Salnikov, Alexei V; Zentgraf, Hanswalter; Büchler, Markus W; Herr, Ingrid

    2012-07-01

    Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide. PMID:22262369

  16. Suppression of autophagy dysregulates the antioxidant response and causes premature senescence of melanocytes.

    Science.gov (United States)

    Zhang, Cheng-Feng; Gruber, Florian; Ni, Chunya; Mildner, Michael; Koenig, Ulrich; Karner, Susanne; Barresi, Caterina; Rossiter, Heidemarie; Narzt, Marie-Sophie; Nagelreiter, Ionela M; Larue, Lionel; Tobin, Desmond J; Eckhart, Leopold; Tschachler, Erwin

    2015-05-01

    Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10-15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs. PMID:25290687

  17. EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells.

    Science.gov (United States)

    Sugita, Shohei; Ito, Kentaro; Yamashiro, Yutaro; Moriya, Shota; Che, Xiao-Fang; Yokoyama, Tomohisa; Hiramoto, Masaki; Miyazawa, Keisuke

    2015-05-22

    Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist. PMID:25858318

  18. Listeriolysin O is necessary and sufficient to induce autophagy during Listeria monocytogenes infection.

    Directory of Open Access Journals (Sweden)

    Nicole Meyer-Morse

    Full Text Available BACKGROUND: Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection. METHODOLOGY/PRINCIPAL FINDINGS: However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs deficient for autophagy (atg5-/-. Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3 lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO, induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5-/- mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5-/- BMDMs. CONCLUSIONS/SIGNIFICANCE: We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.

  19. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  20. Autophagy and intestinal homeostasis.

    Science.gov (United States)

    Patel, Khushbu K; Stappenbeck, Thaddeus S

    2013-01-01

    Nutrient absorption is the basic function that drives mammalian intestinal biology. To facilitate nutrient uptake, the host's epithelial barrier is composed of a single layer of cells. This constraint is problematic, as a design of this type can be easily disrupted. The solution during the course of evolution was to add numerous host defense mechanisms that can help prevent local and systemic infection. These mechanisms include specialized epithelial cells that produce a physiochemical barrier overlying the cellular barrier, robust and organized adaptive and innate immune cells, and the ability to mount an inflammatory response that is commensurate with a specific threat level. The autophagy pathway is a critical cellular process that strongly influences all these functions. Therefore, a fundamental understanding of the components of this pathway and their influence on inflammation, immunity, and barrier function will facilitate our understanding of homeostasis in the gastrointestinal tract. PMID:23216414

  1. Role and Regulation of Autophagy in Heat Stress Responses of Tomato Plants

    Directory of Open Access Journals (Sweden)

    Jie eZhou

    2014-04-01

    Full Text Available As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7 or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.

  2. Autophagy plays an essential role in cigarette smoke-induced expression of MUC5AC in airway epithelium.

    Science.gov (United States)

    Zhou, Jie-Sen; Zhao, Yun; Zhou, Hong-Bin; Wang, Yong; Wu, Yin-Fang; Li, Zhou-Yang; Xuan, Nan-Xia; Zhang, Chao; Hua, Wen; Ying, Song-Min; Li, Wen; Shen, Hua-Hao; Chen, Zhi-Hua

    2016-06-01

    Mucus hypersecretion is a common pathological feature of chronic airway inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, the molecular basis for this condition remains incompletely understood. We have previously demonstrated a critical role of autophagy in COPD pathogenesis through mediating apoptosis of lung epithelial cells. In this study, we aimed to investigate the function of autophagy as well as its upstream and downstream signals in cigarette smoke-induced mucus production in human bronchial epithelial (HBE) cells and in mouse airways. Cigarette smoke extract (CSE), as well as the classical autophagy inducers starvation or Torin-1, significantly triggered MUC5AC expression, and inhibition of autophagy markedly attenuated CSE-induced mucus production. The CSE-induced autophagy was mediated by mitochondrial reactive oxygen species (mitoROS), which regulated mucin expression through the JNK and activator protein-1 pathway. Epidermal growth factor receptor (EGFR) was also required for CSE-induced MUC5AC in HBE cells, but it exerted inconsiderable effects on the autophagy-JNK signaling cascade. Airways of mice with dysfunctional autophagy-related genes displayed a markedly reduced number of goblet cells and attenuated levels of Muc5ac in response to cigarette smoke exposure. These results altogether suggest that mitoROS-dependent autophagy is essential for cigarette smoke-induced mucus hyperproduction in airway epithelial cells, and reemphasize autophagy inhibition as a novel therapeutic strategy for chronic airway diseases. PMID:27036871

  3. Cocaine induces astrocytosis through ER stress-mediated activation of autophagy.

    Science.gov (United States)

    Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

    2016-08-01

    Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation. PMID:27337297

  4. Autophagy and apoptosis: rivals or mates?

    Institute of Scientific and Technical Information of China (English)

    Yan Cheng; Jin-Ming Yang

    2013-01-01

    Autophagy,a cellular process of "self-eating" by which intracellular components are degraded within the lysosome,is an evolutionarily conserved response to various stresses.Autophagy is associated with numerous patho-physiological conditions,and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer.Depending on context,activation of autophagy may promote either cell survival or death,two major events that determine pathological process of many illnesses.Importantly,the activity of autophagy is often associated with apoptosis,another critical cellular process determining cellular fate.A better understanding of biology of autophagy and its implication in human health and disorder,as well as the relationship between autophagy and apoptosis,has the potential of facilitating the development of autophagy-based therapeutic interventions for human diseases such as cancer.

  5. Fluorescence microscopy: A tool to study autophagy

    Science.gov (United States)

    Rai, Shashank; Manjithaya, Ravi

    2015-08-01

    Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

  6. Autophagy and apoptosis: rivals or mates?

    Directory of Open Access Journals (Sweden)

    Yan Cheng

    2013-03-01

    Full Text Available Autophagy, a cellular process of "self-eating" by which intracellular components are degraded within the lysosome, is an evolutionarily conserved response to various stresses. Autophagy is associated with numerous patho-physiological conditions, and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer. Depending on context, activation of autophagy may promote either cell survival or death, two major events that determine pathological process of many illnesses. Importantly, the activity of autophagy is often associated with apoptosis, another critical cellular process determining cellular fate. A better understanding of biology of autophagy and its implication in human health and disorder, as well as the relationship between autophagy and apoptosis, has the potential of facilitating the development of autophagy-based therapeutic interventions for human diseases such as cancer.

  7. Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor

    OpenAIRE

    Torisu, Takehiro; Torisu, Kumiko; Lee, In Hye; Liu, Jie; Malide, Daniela; Combs, Christian A.; Wu, Xufeng S.; Rovira, Ilsa I.; Fergusson, Maria M; Weigert, Roberto; Connelly, Patricia S.; Daniels, Mathew P.; Komatsu, Masaaki; Cao, Liu; Finkel, Toren

    2013-01-01

    Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here, we demonstrate that WPBs are in some cases found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy, or knockdown of the essential autophagy genes Atg5 or Atg7, inhibits the in vitro secretion of VWF. Furthermore, while mice...

  8. Lysosomes and autophagy in aquatic animals.

    Science.gov (United States)

    Moore, Michael N; Kohler, Angela; Lowe, David; Viarengo, Aldo

    2008-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants, as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, autophagic reactions frequently involving reduced lysosomal membrane integrity or stability appear to be effective generic indicators of cellular well-being in eukaryotes: in social amoebae (slime mold), mollusks and fish, autophagy/membrane destabilization is correlated with many stress and toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants can be used for predicting cellular dysfunction and health in aquatic animals, such as shellfish and fish, which are extensively used as sensitive bioindicators in monitoring ecosystem health; and also represent a significant food resource for at least 20% of the global human population. Explanatory frameworks for prediction of pollutant impact on health have been derived encompassing a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. Methods are described for tracking in vivo autophagy of fluorescently labeled cytoplasmic proteins, measuring degradation of radiolabeled intracellular proteins and morphometric measurement of lysosomal/cytoplasmic volume ratio. Additional methods for the determination of lysosomal membrane stability in lower animals are also described, which can be applied to frozen tissue sections, protozoans and isolated cells in vivo. Experimental and simulated results have also indicated that nutritional deprivation (analogous in marine mussels to caloric restriction)-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and simulation modelling is proposed as a practical toolbox for predicting toxic

  9. Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus.

    Science.gov (United States)

    Lee, Lucy E; Witter, R L; Reddy, S M; Wu, P; Yanagida, N; Yoshida, S

    2003-01-01

    Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement. PMID:14562881

  10. STAT3-Mediated Autophagy Dependence Identifies Subtypes of Breast Cancer where Autophagy Inhibition can be Efficacious

    OpenAIRE

    Maycotte, Paola; Gearheart, Christy M.; Barnard, Rebecca; Aryal, Suraj; Mulcahy Levy, Jean M.; Fosmire, Susan P.; Hansen, Ryan J.; Morgan, Michael J.; Christopher C Porter; Gustafson, Daniel L.; Thorburn, Andrew

    2014-01-01

    Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition especially in combination with other therapy could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for...

  11. Involvement of autophagy upregulation in 3,4-methylenedioxymethamphetamine ('ecstasy')-induced serotonergic neurotoxicity.

    Science.gov (United States)

    Li, I-Hsun; Ma, Kuo-Hsing; Kao, Tzu-Jen; Lin, Yang-Yi; Weng, Shao-Ju; Yen, Ting-Yin; Chen, Lih-Chi; Huang, Yuahn-Sieh

    2016-01-01

    It has been suggested that autophagy plays pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug that causes long-term serotonergic neurotoxicity in the brain. Apoptosis and necrosis have been implicated in MDMA-induced neurotoxicity, but the role of autophagy in MDMA-elicited serotonergic toxicity has not been investigated. The present study aimed to examine the contribution of autophagy to neurotoxicity in serotonergic neurons in in vitro and in vivo animal models challenged with MDMA. Here, we demonstrated that in cultured rat serotonergic neurons, MDMA exposure induced LC3B-densely stained autophagosome formation, accompanying by a decrease in neurite outgrowth. Autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated MDMA-induced autophagosome accumulation, and ameliorated MDMA-triggered serotonergic neurite damage and neuron death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in serotonergic neurons and aggravated neurite degeneration. In addition, MDMA-induced autophagy activation in cultured serotonergic neurons might be mediated by serotonin transporter (SERT). In an in vivo animal model administered MDMA, neuroimaging showed that 3-MA protected the serotonin system against MDMA-induced downregulation of SERT evaluated by animal-PET with 4-[(18)F]-ADAM, a SERT radioligand. Taken together, our results demonstrated that MDMA triggers upregulation of autophagy in serotonergic neurons, which appears to be detrimental to neuronal growth. PMID:26610922

  12. AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects.

    Science.gov (United States)

    Papp, Diána; Kovács, Tibor; Billes, Viktor; Varga, Máté; Tarnóci, Anna; Hackler, László; Puskás, László G; Liliom, Hanna; Tárnok, Krisztián; Schlett, Katalin; Borsy, Adrienn; Pádár, Zsolt; Kovács, Attila L; Hegedűs, Krisztina; Juhász, Gábor; Komlós, Marcell; Erdős, Attila; Gulyás, Balázs; Vellai, Tibor

    2016-01-01

    Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases. PMID:26312549

  13. Autophagy and mitophagy in cellular damage control

    Directory of Open Access Journals (Sweden)

    Jianhua Zhang

    2013-01-01

    Full Text Available Autophagy and mitophagy are important cellular processes that are responsible for breaking down cellular contents, preserving energy and safeguarding against accumulation of damaged and aggregated biomolecules. This graphic review gives a broad summary of autophagy and discusses examples where autophagy is important in controlling protein degradation. In addition we highlight how autophagy and mitophagy are involved in the cellular responses to reactive species and mitochondrial dysfunction. The key signaling pathways for mitophagy are described in the context of bioenergetic dysfunction.

  14. Hepatic autophagy contributes to the metabolic response to dietary protein restriction.

    Science.gov (United States)

    Henagan, Tara M; Laeger, Thomas; Navard, Alexandra M; Albarado, Diana; Noland, Robert C; Stadler, Krisztian; Elks, Carrie M; Burk, David; Morrison, Christopher D

    2016-06-01

    Autophagy is an essential cellular response which acts to release stored cellular substrates during nutrient restriction, and particularly plays a key role in the cellular response to amino acid restriction. However, there has been limited work testing whether the induction of autophagy is required for adaptive metabolic responses to dietary protein restriction in the whole animal. Here, we found that moderate dietary protein restriction led to a series of metabolic changes in rats, including increases in food intake and energy expenditure, the downregulation of hepatic fatty acid synthesis gene expression and reduced markers of hepatic mitochondrial number. Importantly, these effects were also associated with an induction of hepatic autophagy. To determine if the induction of autophagy contributes to these metabolic effects, we tested the metabolic response to dietary protein restriction in BCL2-AAA mice, which bear a genetic mutation that impairs autophagy induction. Interestingly, BCL2-AAA mice exhibit exaggerated responses in terms of both food intake and energy expenditure, whereas the effects of protein restriction on hepatic metabolism were significantly blunted. These data demonstrate that restriction of dietary protein is sufficient to trigger hepatic autophagy, and that disruption of autophagy significantly alters both hepatic and whole animal metabolic response to dietary protein restriction. PMID:27173459

  15. p53: The Janus of autophagy?

    OpenAIRE

    Levine, Beth; Abrams, John

    2008-01-01

    The autophagy pathway functions in adaptation to nutrient stress and tumour suppression. The p53 tumour suppressor, previously thought to positively regulate autophagy, may also inhibit it. This dual interplay between p53 and autophagy regulation is enigmatic, but may underlie key aspects of metabolism and cancer biology.

  16. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

    OpenAIRE

    Godar, Rebecca J.; Ma, Xiucui; Liu, Haiyan; Murphy, John T.; Carla J Weinheimer; Kovacs, Attila; Seth D Crosby; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fe...

  17. Protection of Mice from Lethal Endotoxemia by Chimeric Human BPI-Fcγ1 Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    Chen Li; Jing Li; Zhe Lv; Xinghua Guo; Qinghua Chen; Qingli Kong; Yunqing An

    2006-01-01

    To evaluate the potentiality of applying gene therapy to endotoxemia in high-risk patients, we investigated the effects of transferring an adeno-associated virus serotype 2 (AAV2)-mediated BPI-Fcγ1 gene on protecting mice from challenge of lethal endotoxin. The chimeric BPI-Fcγ1 gene consists of two parts, one encods functional N-terminus (1 to 199 amino acidic residues) of human BPI, which is a bactericidal/permeability-increasing protein,and the other encodes Fc segment of human immunoglobulin G1 (Fcγ1). Our results indicated that the target protein could be expressed and secreted into the serum of the gene-transferred mice. After lethal endotoxin challenge, the levels of endotoxin and TNF-α in the gene-transferred mice were decreased. The survival rate of the BPI-Fcγ1 gene-transferred mice was markedly increased. Our data suggest that AAV2-mediated chimeric BPI-Fcγ1 gene delivery can potentially be used clinically for the protection and treatment of endotoxemia and endotoxic shock in high-risk individuals.

  18. ER stress, autophagy, and RNA viruses

    Directory of Open Access Journals (Sweden)

    Jia-Rong eJheng

    2014-08-01

    Full Text Available Endoplasmic reticulum (ER stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR, which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell’s response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host’s defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.

  19. Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux.

    Science.gov (United States)

    Gu, Hong-Feng; Li, Hai-Zhe; Tang, Ya-Ling; Tang, Xiao-Qing; Zheng, Xi-Long; Liao, Duan-Fang

    2016-01-01

    Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis. PMID:27128486

  20. Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xuchen Cao; Bowen Liu; Wenfeng Cao; Weiran Zhang; Fei Zhang; Hongmeng Zhao; Ran Meng

    2013-01-01

    Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables.The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated.Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays.Flow cytometry,fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy,and the role of autophagy was assessed using autophagy inhibitors.Apigenin dose-and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines.The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3,PARP cleavage and Bax/Bcl-2 ratios.The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis.In addition,the apigenin-treated cells exhibited autophagy,as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs)by flow cytometry.Furthermore,the results of the Western blot analysis revealed that the level of LC3-Ⅱ,the processed form of LC3-Ⅰ,was increased.Treatment with the autophagy inhibitor,3-methyladenine (3-MA),significantly enhanced the apoptosis induced by apigenin,which was accompanied by an increase in the level of PARP cleavage.Similar results were also confirmed by flow cytometry and fluorescence microscopy.These results indicate that apigenin has apoptosis-and autophagy-inducing effects in breast cancer cells.Autophagy plays a cyto-protective role in apigenin-induced apoptosis,and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

  1. Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux

    Science.gov (United States)

    Gu, Hong-Feng; Li, Hai-Zhe; Tang, Ya-Ling; Tang, Xiao-Qing; Zheng, Xi-Long; Liao, Duan-Fang

    2016-01-01

    Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis. PMID:27128486

  2. Protective Effect of Gwakhyangjeonggisan Herbal Acupuncture Solution in Glioblastoma Cells: Microarray Analysis of Gene Expression

    Directory of Open Access Journals (Sweden)

    Hong-Seok Lee

    2005-12-01

    Full Text Available Objectives : Neurological disorders have been one of main therapeutic targets of acupuncture. The present study investigated the protective effects of Gwakhyangjeonggisan herbal acupuncture solution (GHAS. Methods : We performed 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay in glioblastoma cells, and did microarray analysis with cells exposed to reactive oxigen species (ROS of hydrogen peroxide by 8.0 k Human cDNA, with cut-off level of 2-fold changes in gene expression. Results : MTT assay showed protective effect of GHAS on the glioblastoma cells exposed to hydrogen peroxide. When glioblastoma cells were exposed to hydrogen peroxide, 24 genes were downregulated. When the cells were pretreated with GHAS before exposure to hydrogen peroxide, 46 genes were downregulated. Many of the genes downregulated by hydrogen peroxide stimulation were decreased in the amount of downregulation or reversed to upregulation. Conclusions : The gene expression changes observed in the present study are supposed to be related to the protective molecular mechanism of GHAS in the glioblastoma cells exposed to ROS stress.

  3. p53 status determines the role of autophagy in pancreatic tumour development

    Science.gov (United States)

    Rosenfeldt, Mathias T.; O'Prey, Jim; Morton, Jennifer P.; Nixon, Colin; Mackay, Gillian; Mrowinska, Agata; Au, Amy; Rai, Taranjit Singh; Zheng, Liang; Ridgway, Rachel; Adams, Peter D.; Anderson, Kurt I.; Gottlieb, Eyal; Sansom, Owen J.; Ryan, Kevin M.

    2013-12-01

    Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)--the most common mutational event in PDAC--develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

  4. Autophagy in 5-Fluorouracil Therapy in Gastrointestinal Cancer: Trends and Challenges

    Institute of Scientific and Technical Information of China (English)

    Jia-Cheng Tang; Yi-Li Feng; Xiao Liang; Xiu-Jun Cai

    2016-01-01

    Objective: 5-Fluorouracil (5-FU)-based combination therapies are standard treatments for gastrointestinal cancer, where the modulation of autophagy is becoming increasingly important in offering effective treatment for patients in clinical practice.This review focuses on the role of autophagy in 5-FU-induced tumor suppression and cancer therapy in the digestive system.Data Sources: All articles published in English from 1996 to date those assess the synergistic effect ofautophagy and 5-FU in gastrointestinal cancer therapy were identified through a systematic online search by use of PubMed.The search terms were "autophagy" and "5-FU" and ("colorectal cancer" or"hepatocellular carcinoma" or"pancreatic adenocarcinoma" or"esophageal cancer" or"gallbladder carcinoma" or "gastric cancer").Study Selection: Critical reviews on relevant aspects and original articles reporting in vitro and/or in vivo results regarding the efficiency ofautophagy and 5-FU in gastrointestinal cancer therapy were reviewed, analyzed, and summarized.The exclusion criteria for the articles were as follows: (1) new materials (e.g., nanomaterial)-induced autophagy;(2) clinical and experimental studies on diagnostic and/or prognostic biomarkers in digestive system cancers;and (3) immunogenic cell death for anticancer chemotherapy.Results: Most cell and animal experiments showed inhibition ofautophagy by either pharmacological approaches or via genetic silencing of autophagy regulatory gene, resulting in a promotion of 5-FU-induced cancer cells death.Meanwhile, autophagy also plays a pro-death role and may mediate cell death in certain cancer cells where apoptosis is defective or difficult to induce.The dual role of autophagy complicates the use of autophagy inhibitor or inducer in cancer chemotherapy and generates inconsistency to an extent in clinic trials.Conclusion: Autophagy might be a therapeutic target that sensitizes the 5-FU treatment in gastrointestinal cancer.

  5. Telemetric control of peripheral lipophagy by hypothalamic autophagy.

    Science.gov (United States)

    Martinez-Lopez, Nuria; Singh, Rajat

    2016-08-01

    Autophagy maintains cellular quality control by degrading organelles, and cytosolic proteins and their aggregates in lysosomes. Autophagy also degrades lipid droplets (LD) through a process termed lipophagy. During lipophagy, LD are sequestered within autophagosomes and degraded by lysosomal acid lipases to generate free fatty acids that are β-oxidized for energy. Lipophagy was discovered in hepatocytes, and since then has been shown to function in diverse cell types. Whether lipophagy degrades LD in the major fat storing cell-the adipocyte-remained unclear. We have found that blocking autophagy in brown adipose tissues (BAT) by deleting the autophagy gene Atg7 in BAT MYF5 (myogenic factor 5)-positive progenitors increases basal lipid content in BAT and decreases lipid utilization during cold exposure-indicating that lipophagy contributes to lipohomeostasis in the adipose tissue. Surprisingly, knocking out Atg7 in hypothalamic proopiomelanocortin (POMC) neurons also blocks lipophagy in BAT and liver suggesting that specific neurons within the central nervous system (CNS) exert telemetric control over lipophagy in BAT and liver. PMID:27341145

  6. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

    LENUS (Irish Health Repository)

    Orfali, Nina

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

  7. The cellular decision between apoptosis and autophagy

    Directory of Open Access Journals (Sweden)

    Yong-Jun Fan

    2013-03-01

    Full Text Available Apoptosis and autophagy are important molecular processes that maintain organismal and cellular homeostasis, respectively. While apoptosis fulfills its role through dismantling damaged or unwanted cells, autophagy maintains cellular homeostasis through recycling selective intracellular organelles and molecules. Yet in some conditions, autophagy can lead to cell death. Apoptosis and autophagy can be stimulated by the same stresses. Emerging evidence indicates an interplay between the core proteins in both pathways, which underlies the molecular mechanism of the crosstalk between apoptosis and autophagy. This review summarizes recent literature on molecules that regulate both the apoptotic and autophagic processes.

  8. Impaired Autophagy in Adult Bone Marrow CD34+ Cells of Patients with Aplastic Anemia: Possible Pathogenic Significance

    Science.gov (United States)

    Huang, Jinbo; Ge, Meili; Lu, Shihong; Shi, Jun; Yu, Wei; Li, Xingxin; Wang, Min; Zhang, Jizhou; Feng, Sizhou; Dong, Shuxu; Cheng, Xuelian; Zheng, Yizhou

    2016-01-01

    Aplastic anemia (AA) is a bone marrow failure syndrome that is caused largely by profound quantitative and qualitative defects of hematopoietic stem and progenitor cells. However, the mechanisms underlying these defects remain unclear. Under conditions of stress, autophagy acts as a protective mechanism for cells. We therefore postulated that autophagy in CD34+ hematopoietic progenitor cells (HPCs) from AA patients might be impaired and play a role in the pathogenesis of AA. To test this hypothesis, we tested autophagy in CD34+ cells from AA samples and healthy controls and investigated the effect of autophagy on the survival of adult human bone marrow CD34+ cells. We found that the level of autophagy in CD34+ cells from AA patients was significantly lower than in age/sex-matched healthy controls, and lower in cases of severe AA than in those with non-severe AA. Autophagy in CD34+ cells improved upon amelioration of AA but, compared to healthy controls, was still significantly reduced even in AA patients who had achieved a complete, long-term response. We also showed that although the basal autophagy in CD34+ cells was low, the autophagic response of CD34+ cells to “adversity” was rapid. Finally, impaired autophagy resulted in reduced differentiation and proliferation of CD34+ cells and sensitized them to death and apoptosis. Thus, our results confirm that autophagy in CD34+ cells from AA patients is impaired, that autophagy is required for the survival of CD34+ cells, and that impaired autophagy in CD34+ HPCs may play an important role in the pathogenesis of AA. PMID:26930650

  9. Advances in Autophagy Regulatory Mechanisms

    Directory of Open Access Journals (Sweden)

    Laura E. Gallagher

    2016-05-01

    Full Text Available Autophagy plays a critical role in cell metabolism by degrading and recycling internal components when challenged with limited nutrients. This fundamental and conserved mechanism is based on a membrane trafficking pathway in which nascent autophagosomes engulf cytoplasmic cargo to form vesicles that transport their content to the lysosome for degradation. Based on this simple scheme, autophagy modulates cellular metabolism and cytoplasmic quality control to influence an unexpectedly wide range of normal mammalian physiology and pathophysiology. In this review, we summarise recent advancements in three broad areas of autophagy regulation. We discuss current models on how autophagosomes are initiated from endogenous membranes. We detail how the uncoordinated 51-like kinase (ULK complex becomes activated downstream of mechanistic target of rapamycin complex 1 (MTORC1. Finally, we summarise the upstream signalling mechanisms that can sense amino acid availability leading to activation of MTORC1.

  10. Advances in Autophagy Regulatory Mechanisms.

    Science.gov (United States)

    Gallagher, Laura E; Williamson, Leon E; Chan, Edmond Y W

    2016-01-01

    Autophagy plays a critical role in cell metabolism by degrading and recycling internal components when challenged with limited nutrients. This fundamental and conserved mechanism is based on a membrane trafficking pathway in which nascent autophagosomes engulf cytoplasmic cargo to form vesicles that transport their content to the lysosome for degradation. Based on this simple scheme, autophagy modulates cellular metabolism and cytoplasmic quality control to influence an unexpectedly wide range of normal mammalian physiology and pathophysiology. In this review, we summarise recent advancements in three broad areas of autophagy regulation. We discuss current models on how autophagosomes are initiated from endogenous membranes. We detail how the uncoordinated 51-like kinase (ULK) complex becomes activated downstream of mechanistic target of rapamycin complex 1 (MTORC1). Finally, we summarise the upstream signalling mechanisms that can sense amino acid availability leading to activation of MTORC1. PMID:27187479

  11. Correlative Gene Expression to Protective Seroconversion in Rift Valley Fever Vaccinates.

    Science.gov (United States)

    Laughlin, Richard C; Drake, Kenneth L; Morrill, John C; Adams, L Garry

    2016-01-01

    Rift Valley fever Virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease, Rift Valley fever (RVF). In both humans and livestock, protective immunity can be achieved through vaccination. Earlier and more recent vaccine trials in cattle and sheep demonstrated a strong neutralizing antibody and total IgG response induced by the RVF vaccine, authentic recombinant MP-12 (arMP-12). From previous work, protective immunity in sheep and cattle vaccinates normally occurs from 7 to 21 days after inoculation with arMP-12. While the serology and protective response induced by arMP-12 has been studied, little attention has been paid to the underlying molecular and genetic events occurring prior to the serologic immune response. To address this, we isolated RNA from whole blood of vaccinated calves over a time course of 21 days before and after vaccination with arMP-12. The time course RNAs were sequenced by RNASeq and bioinformatically analyzed. Our results revealed time-dependent activation or repression of numerous gene ontologies and pathways related to the vaccine induced immune response and its regulation. Additional bioinformatic analyses identified a correlative relationship between specific host immune response genes and protective immunity prior to the detection of protective serum neutralizing antibody responses. These results contribute an important proof of concept for identifying molecular and genetic components underlying the immune response to RVF vaccination and protection prior to serologic detection. PMID:26783758

  12. Intestinal Epithelium and Autophagy: Partners in Gut Homeostasis

    OpenAIRE

    Randall-Demllo, Sarron; Chieppa, Marcello; Eri, Rajaraman

    2013-01-01

    One of the most significant challenges of cell biology is to understand how each type of cell copes with its specific workload without suffering damage. Among the most intriguing questions concerns intestinal epithelial cells in mammals; these cells act as a barrier between the internally protected region and the external environment that is exposed constantly to food and microbes. A major process involved in the processing of microbes is autophagy. In the intestine, through multiple, complex...

  13. Transcription factors and cognate signalling cascades in the regulation of autophagy.

    Science.gov (United States)

    Chandra, Vemika; Bhagyaraj, Ella; Parkesh, Raman; Gupta, Pawan

    2016-05-01

    Autophagy is a process that maintains the equilibrium between biosynthesis and the recycling of cellular constituents; it is critical for avoiding the pathophysiology that results from imbalance in cellular homeostasis. Recent reports indicate the need for the design of high-throughput screening assays to identify targets and small molecules for autophagy modulation. For such screening, however, a better understanding of the regulation of autophagy is essential. In addition to regulation by various signalling cascades, regulation of gene expression by transcription factors is also critical. This review focuses on the various transcription factors as well as the corresponding signalling molecules that act together to translate the stimuli to effector molecules that up- or downregulate autophagy. This review rationalizes the importance of these transcription factors functioning in tandem with cognate signalling molecules and their interfaces as possible therapeutic targets for more specific pharmacological interventions. PMID:25651938

  14. Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy.

    Science.gov (United States)

    Heintze, Jacob; Costa, Joana R; Weber, Melanie; Ketteler, Robin

    2016-09-01

    Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling. PMID:27328773

  15. Host-induced silencing of Fusarium culmorum genes protects wheat from infection.

    Science.gov (United States)

    Chen, Wanxin; Kastner, Christine; Nowara, Daniela; Oliveira-Garcia, Ely; Rutten, Twan; Zhao, Yusheng; Deising, Holger B; Kumlehn, Jochen; Schweizer, Patrick

    2016-09-01

    Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant-fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the β-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes. PMID:27540093

  16. Modulation of pathogen recognition by autophagy

    Directory of Open Access Journals (Sweden)

    Ji Eun eOh

    2012-03-01

    Full Text Available Autophagy is an ancient biological process for maintaining cellular homeostasis by degradation of long-lived cytosolic proteins and organelles. Recent studies demonstrated that autophagy is availed by immune cells to regulate innate immunity. On the one hand, cells exert direct effector function by degrading intracellular pathogens; on the other hand, autophagy modulates pathogen recognition and downstream signaling for innate immune responses. Pathogen recognition via pattern recognition receptors induces autophagy. The function of phagocytic cells is enhanced by recruitment of autophagy-related proteins. Moreover, autophagy acts as a delivery system for viral replication complexes to migrate to the endosomal compartments where virus sensing occurs. In another case, key molecules of the autophagic pathway have been found to negatively regulate immune signaling, thus preventing aberrant activation of cytokine production and consequent immune responses. In this review, we focus on the recent advances in the role of autophagy in pathogen recognition and modulation of innate immune responses.

  17. Historical landmarks of autophagy research

    OpenAIRE

    Ohsumi, Yoshinori

    2013-01-01

    The year of 2013 marked the 50th anniversary of C de Duve's coining of the term “autophagy” for the degradation process of cytoplasmic constituents in the lysosome/vacuole. This year we regretfully lost this great scientist, who contributed much during the early years of research to the field of autophagy. Soon after the discovery of lysosomes by de Duve, electron microscopy revealed autophagy as a means of delivering intracellular components to the lysosome. For a long time after the discove...

  18. Hepatic gene expression profiling reveals protective responses in Atlantic salmon vaccinated against furunculosis

    Directory of Open Access Journals (Sweden)

    Jørgensen Sven

    2009-10-01

    Full Text Available Abstract Background Furunculosis, a disease caused with gram negative bacteria Aeromonas salmonicida produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon. Results Naïve and vaccinated fish were challenged by co-habitance. Fish with symptoms of furunculosis at the onset of mass mortality (LR - low resistance and survivors (HR - high resistance were sampled. Hepatic gene expression was analyzed with microarray (SFA2.0 - immunochip and real-time qPCR. Comparison of LR and HR indicated changes associated with the protection and results obtained with naïve fish were used to find and filter the vaccine-independent responses. Genes involved in recruitment and migration of immune cells changed expression in both directions with greater magnitude in LR. Induction of the regulators of immune responses was either equal (NFkB or greater (Jun in LR. Expression levels of proteasome components and extracellular proteases were higher in LR while protease inhibitors were up-regulated in HR. Differences in chaperones and protein adaptors, scavengers of reactive oxygen species and genes for proteins of iron metabolism suggested cellular and oxidative stress in LR. Reduced levels of free iron and heme can be predicted in LR by gene expression profiles with no protection against pathogen. The level of complement regulation was greater in HR, which showed up-regulation of the components of membrane attack complex and the complement proteins that protect the host against the auto-immune damages. HR fish was also characterized with up-regulation of genes for proteins involved in the protection of extracellular matrix, lipid metabolism and clearance of endogenous and

  19. Hepatic gene expression profiling reveals protective responses in Atlantic salmon vaccinated against furunculosis

    Science.gov (United States)

    Škugor, Stanko; Jørgensen, Sven Martin; Gjerde, Bjarne; Krasnov, Aleksei

    2009-01-01

    Background Furunculosis, a disease caused with gram negative bacteria Aeromonas salmonicida produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon. Results Naïve and vaccinated fish were challenged by co-habitance. Fish with symptoms of furunculosis at the onset of mass mortality (LR - low resistance) and survivors (HR - high resistance) were sampled. Hepatic gene expression was analyzed with microarray (SFA2.0 - immunochip) and real-time qPCR. Comparison of LR and HR indicated changes associated with the protection and results obtained with naïve fish were used to find and filter the vaccine-independent responses. Genes involved in recruitment and migration of immune cells changed expression in both directions with greater magnitude in LR. Induction of the regulators of immune responses was either equal (NFkB) or greater (Jun) in LR. Expression levels of proteasome components and extracellular proteases were higher in LR while protease inhibitors were up-regulated in HR. Differences in chaperones and protein adaptors, scavengers of reactive oxygen species and genes for proteins of iron metabolism suggested cellular and oxidative stress in LR. Reduced levels of free iron and heme can be predicted in LR by gene expression profiles with no protection against pathogen. The level of complement regulation was greater in HR, which showed up-regulation of the components of membrane attack complex and the complement proteins that protect the host against the auto-immune damages. HR fish was also characterized with up-regulation of genes for proteins involved in the protection of extracellular matrix, lipid metabolism and clearance of endogenous and exogenous toxic compounds

  20. Liraglutide prevents high glucose level induced insulinoma cells apoptosis by targeting autophagy

    Institute of Scientific and Technical Information of China (English)

    CHEN Ze-fang; LI Yan-bo; HAN Jun-yong; YIN Jia-jing; WANG Yang; ZHU Li-bo; XIE Guang-ying

    2013-01-01

    Background The pathophysiology of type 2 diabetes is progressive pancreatic beta cell failure with consequential reduced insulin secretion.Glucotoxicity results in the reduction of beta cell mass in type 2 diabetes by inducing apoptosis.Autophagy is essential for the maintenance of normal islet architecture and plays a crucial role in maintaining the intracellular insulin content by accelerating the insulin degradation rate in beta cells.Recently more attention has been paid to the effect of autophagy in type 2 diabetes.The regulatory pathway of autophagy in controlling pancreatic beta cells is still not clear.The aim of our study was to evaluate whether liraglutide can inhibit apoptosis and modulate autophagy in vitro in insulinoma cells (INS-1 cells).Methods INS-1 cells were incubated for 24 hours in the presence or absence of high levels of glucose,liraglutide (a long-acting human glucagon-like peptide-1 analogue),or 3-methyadenine (3-MA).Cell viability was measured using the Cell Counting Kit-8 (CCK8) viability assay.Autophagy of INS-1 cells was tested by monodansylcadaverine (MDC)staining,an autophagy fluorescent compound used for the labeling of autophagic vacuoles,and by Western blotting of microtubule-associated protein I light chain 3 (LC3),a biochemical markers of autophagic initiation.Results The viability of INS-1 cells was reduced after treatment with high levels of glucose.The viability of INS-1 cells was reduced and apoptosis was increased when autophagy was inhibited.The viability of INS-1 cells was significantly increased by adding liraglutide to supplement high glucose level medium compared with the cells treated with high glucose levels alone.Conclusions Apoptosis and autophagy were increased in rat INS-1 cells when treated with high level of glucose,and the viability of INS-1 cells was significantly reduced by inhibiting autophagy.Liraglutide protected INS-1 cells from high glucose level-induced apoptosis that is accompanied by a significant

  1. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  2. miR-224-3p inhibits autophagy in cervical cancer cells by targeting FIP200.

    Science.gov (United States)

    Fang, Wang; Shu, Shan; Yongmei, Li; Endong, Zhu; Lirong, Yin; Bei, Sun

    2016-01-01

    Cervical cancer (CC) is a malignant solid tumor, which is one of the main causes of morbidity and mortality in women. Persistent High-risk human papillomavirus (hrHPV) infection is closely related to cervical cancer and autophagy has been suggested to inhibit viral infections. miRNAs have been reported to regulate autophagy in many solid tumors with many studies implicating miR-224-3p in the regulation of autophagy. In this study, we performed a miRNA microarray analysis on CC tissues and found that a large number of miRNAs with differential expressions in hrHPV-infected tissues. We identified miR-224-3p as a candidate miRNA selectively up regulated in HPV-infected tissues and cell lines. Further analysis revealed that miR-224-3p regulates autophagy in cervical cancer tissues and cell lines. While the overexpression of miR-224-3p inhibits autophagy in HPV-infected cells, knocking down endogenous miR-224-3p increases autophagy activity in the same cells. In addition, we found that miR-224-3p directly inhibits the expression of autophagy related gene, FAK family-interacting protein of 200 kDa (FIP200). In summary, we found that miR-224-3p regulates autophagy in hrHPV-induced cervical cancer cells through targeting FIP200 expression. PMID:27615604

  3. The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides general stress protection.

    Science.gov (United States)

    Rodríguez-Rojas, Alexandro; Blázquez, Jesús

    2009-02-01

    Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H(2)O(2)-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation. PMID:19028889

  4. Noncoding RNA blockade of autophagy is therapeutic in medullary thyroid cancer

    International Nuclear Information System (INIS)

    Micro-RNAs are dysregulated in medullary thyroid carcinoma (MTC) and preliminary studies have shown that miRNAs may enact a therapeutic effect through changes in autophagic flux. Our aim was to study the in vitro effect of miR-9-3p on MTC cell viability, autophagy and to investigate the mRNA autophagy gene profile of sporadic versus hereditary MTC. The therapeutic role of miR-9-3p was investigated in vitro using human MTC cell lines (TT and MZ-CRC-1 cells), cell viability assays, and functional mechanism studies with a focus on cell cycle, apoptosis, and autophagy. Post-miR-9-3p transfection mRNA profiling of cell lines was performed using a customized, quantitative RT-PCR gene array card. This card was also run on clinical tumor samples (sporadic: n = 6; hereditary: n = 6) and correlated with clinical data. Mir-9-3p transfection resulted in reduced in vitro cell viability; an effect mediated through autophagy inhibition. This was accompanied by evidence of G2 arrest in the TT cell line and increased apoptosis in both cell lines. Atg5 was validated as a predicted miR-9-3p mRNA target in TT cells. Post-miR-9-3p transfection array studies showed a significant global decline in autophagy gene expression (most notably in PIK3C3, mTOR, and LAMP-1). Autophagy gene mRNAs were generally overexpressed in sporadic (vs. hereditary MTC) and Beclin-1 overexpression was shown to correlate with residual disease. Autophagy is a tumor cell survival mechanism in MTC that when disabled, is of therapeutic advantage. Beclin-1 expression may be a useful prognostic biomarker of aggressive disease

  5. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    Science.gov (United States)

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

  6. Ordered bulk degradation via autophagy

    DEFF Research Database (Denmark)

    Dengjel, Jörn; Kristensen, Anders Riis; Andersen, Jens S

    2008-01-01

    proteasomal and lysosomal degradation ample cross-talk between the two degradation pathways became evident. Degradation via autophagy appeared to be ordered and regulated at the protein complex/organelle level. This raises several important questions such as: can macroautophagy itself be specific and what is...

  7. Protection and synergism by recombinant fowl pox vaccines expressing genes from Marek's disease virus.

    Science.gov (United States)

    Nazerian, K; Witter, R L; Lee, L F; Yanagida, N

    1996-01-01

    Recombinant fowl poxviruses (rFPV) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B (gB1), C (gC), and D (gD) and tegument proteins UL47 and UL48, as well as genes from serotypes 2 and 3 MDV coding for glycoprotein B (gB2 and gB3). These rFPVs, alone and in various combinations, including combinations of fowl poxvirus (FPV)/gBs with turkey herpesvirus (HVT), were evaluated for ability to protect maternal antibody-positive (ab+) and -negative (ab-) chickens against challenge with highly virulent MDV isolates. The protective efficacy was also compared with that of prototype Marek's disease (MD) vaccines. No protection was induced in ab+ chickens by rFPV expressing gC, gD, UL47, or UL48. In contrast, the rFPV/gB1 construct protected about 23% of ab+ chickens against MDV challenge compared with 26% for cell-associated HVT. Levels of protection by rFPV/gBs of different MDV serotypes was highest for gB1, intermediate for gB2, and lowest for gB3. When rFPV/gB1 was combined with cell-associated HVT, protection was enhanced by an average of 138% compared with the best component monovalent vaccine, and the mean level of protection was 59% compared with 67% for the HVT+SB-1 bivalent vaccine. Relatively high protection (50%) and enhancement (200%) were also observed between rFPV/gB1 and cell-free HVT. These results suggest a specific synergistic interaction between rFPV/gB1 and HVT, possibly analogous to that previously described between serotypes 2 and 3 viruses. Levels of protection by rFPV/ gB1 alone or by bivalent rFPV/gB1+cell-associated HVT were similar to those of conventional cell-associated MD vaccines. However, the bivalent rFPV/gB1+cell-free HVT vaccine was clearly more protective than cell-free HVT alone and, thus, may be the most protective, entirely cell-free MD vaccine thus far described. PMID:8790888

  8. Hepatic gene expression profiling reveals protective responses in Atlantic salmon vaccinated against furunculosis

    OpenAIRE

    Jørgensen Sven; Škugor Stanko; Gjerde Bjarne; Krasnov Aleksei

    2009-01-01

    Abstract Background Furunculosis, a disease caused with gram negative bacteria Aeromonas salmonicida produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon. Results Naïve and vaccinated fish were...

  9. Inhibition of autophagy ameliorates pulmonary microvascular dilation and PMVECs excessive proliferation in rat experimental hepatopulmonary syndrome

    Science.gov (United States)

    Xu, Duo; Chen, Bing; Gu, Jianteng; Chen, Lin; Belguise, Karine; Wang, Xiaobo; Yi, Bin; Lu, Kaizhi

    2016-01-01

    Hepatopulmonary syndrome (HPS) is a defective liver-induced pulmonary vascular disorder with massive pulmonary microvascular dilation and excessive proliferation of pulmonary microvascular endothelial cells (PMVECs). Growing evidence suggests that autophagy is involved in pulmonary diseases, protectively or detrimentally. Thus, it is interesting and important to explore whether autophagy might be involved in and critical in HPS. In the present study, we report that autophagy was activated in common bile duct ligation (CBDL) rats and cultured pulmonary PMVECs induced by CBDL rat serum, two accepted in vivo and in vitro experimental models of HPS. Furthermore, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) significantly alleviated pathological alterations and typical symptom of HPS in CBDL rats in vivo, and consistently 3-MA significantly attenuated the CBDL rat serum-induced excessive proliferation of PMVECs in vitro. All these changes mediated by 3-MA might explain the observed prominent improvement of pulmonary appearance, edema, microvascular dilatation and arterial oxygenation in vivo. Collectively, these results suggest that autophagy activation may play a critical role in the pathogenesis of HPS, and autophagy inhibition may have a therapeutic potential for this disease. PMID:27480323

  10. Quercetin attenuates renal ischemia/reperfusion injury via an activation of AMP-activated protein kinase-regulated autophagy pathway.

    Science.gov (United States)

    Chen, Bo-Lin; Wang, Li-Ting; Huang, Kuo-How; Wang, Ching-Chia; Chiang, Chih-Kang; Liu, Shing-Hwa

    2014-11-01

    Renal ischemia/reperfusion (I/R) is a major cause of acute renal failure. Quercetin, a flavonoid antioxidant, presents in many kinds of food. The molecular mechanism of quercetin on renal protection during I/R is still unclear. Here, we investigated the role of AMP-activated protein kinase (AMPK)-regulated autophagy in renal protection by quercetin. To investigate whether quercetin protects renal cells from I/R-induced cell injury, an in vitro model of I/R and an in vivo I/R model were used. Cell apoptosis was determined by propidium iodide/annexin V staining. Western blotting and immunofluorescence were used to determine the autophagy. AMPK expression was inhibited with appropriate short hairpin RNA (shRNA). In cultured renal tubular cell I/R model, quercetin decreased the cell injury, up-regulated the AMPK phosphorylation, down-regulated the mammalian target of rapamycin (mTOR) phosphorylation and activated autophagy during I/R. Knockdown of AMPK by shRNA transfection decreased the quercetin-induced autophagy but did not affect the mTOR phosphorylation. In I/R mouse model, quercetin decreased the increased serum creatinine level and altered renal histological score. Quercetin also increased AMPK phosphorylation, inhibited the mTOR phosphorylation and activated autophagy in the kidneys of I/R mice. These results suggest that quercetin activates an AMPK-regulated autophagy signaling pathway, which offers a protective effect in renal I/R injury. PMID:25087994

  11. EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy.

    Science.gov (United States)

    Li, Mengtao; Lu, Guang; Hu, Jia; Shen, Xue; Ju, Jiabao; Gao, Yuanxu; Qu, Liujing; Xia, Yan; Chen, Yingyu; Bai, Yun

    2016-03-01

    Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation. PMID:26905199

  12. EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy

    Directory of Open Access Journals (Sweden)

    Mengtao Li

    2016-03-01

    Full Text Available Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation.

  13. Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

    Institute of Scientific and Technical Information of China (English)

    Lu-yang YU; Bo LIN; Zhen-lin ZHANG; Li-he GUO

    2004-01-01

    AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Westem blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

  14. The role of autophagy in Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Yaru Dong; Xiaoheng Xu; Zhong Xu

    2012-01-01

    Although Parkinson's disease is the most common neurodegenerative movement disorder, the mechanisms of pathogenesis remain poorly understood. Recent findings have shown that deregulation of the autophagy-lysosome pathway is involved in the pathogenesis of Parkinson's disease. This review summarizes the most recent findings and discusses the unique role of the autophagy-lysosome pathway in Parkinson's disease to highlight the possibility of Parkinson's disease treatment strategies that incorporate autophagy-lysosome pathway modulation.

  15. Oxidative Stress and Autophagy in Cardiovascular Homeostasis

    OpenAIRE

    Morales, Cyndi R.; Pedrozo, Zully; Lavandero, Sergio; Hill, Joseph A.

    2014-01-01

    Significance: Autophagy is an evolutionarily ancient process of intracellular protein and organelle recycling required to maintain cellular homeostasis in the face of a wide variety of stresses. Dysregulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative damage. Both autophagy and ROS/RNS serve pathological or adaptive roles within cardiomyocytes, depending on the context. Recent Advances: ROS/RNS and autophagy communicate with each other via both tra...

  16. Autophagy and oxidative stress in cardiovascular diseases

    OpenAIRE

    Mei, Yu; Thompson, Melissa D.; Cohen, Richard A.; Tong, XiaoYong

    2014-01-01

    Autophagy is a highly conserved degradation process by which intracellular components, including soluble macromolecules (e.g. nucleic acids, proteins, carbohydrates, and lipids) and dysfunctional organelles (e.g. mitochondria, ribosomes, peroxisomes, and endoplasmic reticulum) are degraded by the lysosome. Autophagy is orchestrated by the autophagy related protein (Atg) composed protein complexes to form autophagosomes, which fuse with lysosomes to generate autolysosomes where the contents ar...

  17. Streptococcus pneumoniae induces autophagy through the inhibition of the PI3K-I/Akt/mTOR pathway and ROS hypergeneration in A549 cells.

    Directory of Open Access Journals (Sweden)

    Pu Li

    Full Text Available The present study focused on the action mechanism of S. pneumoniae (Sp in inducing autophagy in human alveolar epithelial cells. Sp, a gram-positive extracellular bacterium, activates autophagy with considerably increased microtuble-associated protein light chain 3 (LC3 punctation in A549 cells. The accumulation of typical autophagosomes and conjugation of LC3 to phosphatidylethanolamine were observed in Sp-infected cells as an indication of autophagy. Using the pneumolysin (PLY mutant, we successfully demonstrated that PLY is involved in initiating autophagy without affecting the expression levels of PI3K-III and Beclin1. PLY-mediated autophagy depends on the inhibition of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway. Furthermore, Sp could also lead to the reactive oxygen species (ROS hypergeneration in A549 cells. Taken together, Sp infection-induced autophagy is PLY-mediated through ROS hypergeneration and mTOR inhibition. PI3K-I and rapamycin (autophagy inducers enhanced bacterial clearance, whereas wortmannin (autophagy inhibitor and acetylcysteine (ROS inhibitor reduced intracellular bacteria clearance. Thus, Sp-induced autophagy represents a host-protective mechanism, providing new insight into the pathogenesis of respiratory tract Sp infection.

  18. Does the KIR2DS5 gene protect from some human diseases?

    Directory of Open Access Journals (Sweden)

    Izabela Nowak

    Full Text Available BACKGROUND: KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation. METHODOLOGY/PRINCIPAL FINDINGS: In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p=0.003, odds ratio [OR]=0.47, confidence interval [CI]=0.28-0.79, endometriosis (p=0.03, OR=0.25, CI = 0.07-0.82 and acute rejection of kidney graft (p=0.0056, OR=0.44, CI=0.24-0.80, but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05. In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p=0.0003, OR=0.35, CI=0.19-0.65, whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p=0.0017, OR=1.92, CI=1.28-2.89, whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p=0.005, OR=1.47, CI=1.13-1.92. The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p=0.017, OR=0.47, CI=0.25-0.89, whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p=0.0015, OR=2.13, CI=1.34-3.37. CONCLUSIONS/SIGNIFICANCE: Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.

  19. Involvement of Autophagy in Coronavirus Replication

    Directory of Open Access Journals (Sweden)

    Paul Britton

    2012-11-01

    Full Text Available Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3 in coronavirus replication.

  20. Probiotic Lactobacillus rhamnosus GG mono-association suppresses human rotavirus-induced autophagy in the gnotobiotic piglet intestine

    OpenAIRE

    Wu, Shaoping; Yuan, Lijuan; Zhang, Yongguo; Liu, Fangning; Li, Guohua; Wen, Ke; Kocher, Jacob; Yang, Xingdong; Sun, Jun

    2013-01-01

    Abstract Background Human rotavirus (HRV) is the most important cause of severe diarrhea in infants and young children. Probiotic Lactobacillus rhamnosus GG (LGG) reduces rotavirus infection and diarrhea. However, the molecular mechanisms of LGG-mediated protection from rotavirus infection are poorly understood. Autophagy plays an essential role in responses to microbial pathogens. However, the role of autophagy in HRV infection and LGG treatment is unknown. We hypothesize that rotavirus gast...

  1. Autophagy in muscle of glucose-infusion hyperglycemia rats and streptozotocin-induced hyperglycemia rats via selective activation of m-TOR or FoxO3.

    Directory of Open Access Journals (Sweden)

    Pengfei Lv

    Full Text Available Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.

  2. Autophagy in the thymic epithelium is dispensable for the development of self-tolerance in a novel mouse model.

    Directory of Open Access Journals (Sweden)

    Supawadee Sukseree

    Full Text Available The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14, including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre. Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1 in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+ CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.

  3. LAPping up dead cells to prevent lupus nephritis: a novel role for noncanonical autophagy in autoimmunity.

    Science.gov (United States)

    Leventhal, Jeremy S; Ross, Michael J

    2016-08-01

    The mechanisms underlying the development of systemic lupus erythematosus and lupus nephritis remain poorly understood. A recent study demonstrates that deficiencies in the immune system's ability to degrade scavenged dead cells via noncanonical autophagy is sufficient to break immune tolerance and produce features commonly seen in lupus, including circulating autoantibodies, inflammatory cytokines, and nephritis. This work provides a possible mechanism for the association of polymorphisms in autophagy genes with the risk of lupus. PMID:27418084

  4. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

    OpenAIRE

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M.

    2008-01-01

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a r...

  5. Effect of Exendin-4 on Autophagy Clearance in Beta Cell of Rats with Tacrolimus-induced Diabetes Mellitus

    Science.gov (United States)

    Lim, Sun Woo; Jin, Long; Jin, Jian; Yang, Chul Woo

    2016-01-01

    Growing evidence suggests that GLP-1 protects beta cells against various cellular injuries by modulating autophagy. In this study, we examined whether exendin-4 (Ex-4), a GLP-1 analog, had preventive effects on tacrolimus (Tac)-induced beta cell injury by improving autophagy clearance. Rats with Tac-induced diabetes mellitus exhibited increased autophagy-associated protein expression, light chain 3B levels, and autophagic vacuole numbers in pancreatic beta cells. Additionally, Tac increased autophagy in a dose- and time-dependent manner in vitro, and inhibition of autophagosome using 3-methyladenine reduced Tac-induced islet cell injury by decreasing reactive oxygen species production and apoptosis. Ex-4 treatment decreased Tac-induced hyperglycaemia, oxidative stress, and apoptosis, accompanied by decreased autophagy-associated protein expression and autophagosome numbers. In vivo and in vitro studies showed that Tac treatment impaired lysosomal function and autophagosome-lysosome fusion; these processes were improve by Ex-4 treatment. Moreover, addition of bafilomycin A1, an inhibitor of lysosomal function, abolished the protective effects of Ex-4. Our findings reveal that Tac-induced diabetes mellitus was a state of excessive burden of autophagosomes and impairment of autophagy clearance and that Ex-4 protected against Tac-induced pancreatic islet injury by reducing the burden of autophagosomes via activation of autophagosome clearance. Thus, Ex-4 had therapeutic effects on Tac-induced pancreatic beta cell injury. PMID:27436514

  6. Coffee induces autophagy in vivo

    OpenAIRE

    Pietrocola, Federico; Malik, Shoaib Ahmad; Mariño, Guillermo; Vacchelli, Erika; Senovilla, Laura; Chaba, Kariman; Niso-Santano, Mireia; Maiuri, Maria Chiara; Madeo, Frank; Kroemer, Guido

    2014-01-01

    Epidemiological studies and clinical trials revealed that chronic consumption coffee is associated with the inhibition of several metabolic diseases as well as reduction in overall and cause-specific mortality. We show that both natural and decaffeinated brands of coffee similarly rapidly trigger autophagy in mice. One to 4 h after coffee consumption, we observed an increase in autophagic flux in all investigated organs (liver, muscle, heart) in vivo, as indicated by the increased lipidation ...

  7. Protection from radiation injury through oral administration of PF4 gene carried by attenuated salmonella

    International Nuclear Information System (INIS)

    Objective: To investigate the in vivo radiation protection effect of PF4 by oral administration of attenuated salmonella as the carrier in mice. Methods: The eukaryotic vector pIRES2-EGFP-carried PF4 gene was transferred into an aroA-autotrophic mutant of salmonella typhimurium (SL3261), which was administered orally to BALBPc mice at 1x108 PFu once every interval three days. At 12 hours after the third oral administration the mice were subjected to a total body irradiation (TBI) of 700 cGy by a 60Co source. The protective effect of SL3261/PF4 was determined by detection GFP ( green fluorescence protein) expression in tissues, peripheral blood count, culture of bone marrow colony-forming cells and survival time of mice. Results: Expression of GFP could be detected in the liver, spleen, intestine, kidney, peripheral blood and bone marrow. On days 7 and 14 after irradiation, Compared to controls, there were obvious differences in number of bone marrow mononuclear cells, CFU-GM (granulocyte-macrophage colony-forming unit ) and HPP-CFC (high proliferating potential-colony-forming cells) of mice treated with SL3261/PF4 (P<0.05) as well as prolongation of the survival time. Conclusion: These data demonstrate for the first time that PF4 protects mice from TBI injury and accelerates recovery of hematopoiesis by oral administration of attenuated salmonella carrying PF4 gene. (authors)

  8. The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys

    Science.gov (United States)

    Etienne, Lucie; Bibollet-Ruche, Frederic; Sudmant, Peter H.; Wu, Lily I.; Hahn, Beatrice H.; Emerman, Michael

    2015-01-01

    Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee’s main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4+ T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses. PMID:26394054

  9. Autophagy selectivity through receptor clustering

    Science.gov (United States)

    Rutenberg, Andrew; Brown, Aidan

    Substrate selectivity in autophagy requires an all-or-none cellular response. We focus on peroxisomes, for which autophagy receptor proteins NBR1 and p62 are well characterized. Using computational models, we explore the hypothesis that physical clustering of autophagy receptor proteins on the peroxisome surface provides an appropriate all-or-none response. We find that larger peroxisomes nucleate NBR1 clusters first, and lose them due to competitive coarsening last, resulting in significant size-selectivity. We then consider a secondary hypothesis that p62 inhibits NBR1 cluster formation. We find that p62 inhibition enhances size-selectivity enough that, even if there is no change of the pexophagy rate, the volume of remaining peroxisomes can significantly decrease. We find that enhanced ubiquitin levels suppress size-selectivity, and that this effect is more pronounced for individual peroxisomes. Sufficient ubiquitin allows receptor clusters to form on even the smallest peroxisomes. We conclude that NBR1 cluster formation provides a viable physical mechanism for all-or-none substrate selectivity in pexophagy. We predict that cluster formation is associated with significant size-selectivity. Now at Simon Fraser University.

  10. Autophagy and the (prorenin receptor

    Directory of Open Access Journals (Sweden)

    KatrinaJeanBinger

    2013-10-01

    Full Text Available The (prorenin receptor (PRR is a newly reported member of the renin-angiotensin system (RAS; a hormonal cascade responsible for regulating blood pressure. Originally, the identification of PRR was heralded as the next drug target of the RAS, of which such therapies would have increased benefits against target-organ damage and hypertension. However, in the years since its discovery several conditional knockout mouse models of PRR have demonstrated an essential role for this receptor unrelated to the renin-angiotensin system and blood pressure. Deletion of PRR in podocytes or cardiomyocytes resulted in the rapid onset of organ failure, eventuating in animal mortality after only a matter of weeks. In both cases, deletion of PRR resulted in the intracellular accumulation of autophagosomes and misfolded proteins, indicating a disturbance in autophagy. In light of the fact that the majority of PRR is located intracellularly, this molecular function appears to be more relevant than its ability to bind to high, non-physiological concentrations of (prorenin. This review will focus on the role of PRR in autophagy and its importance in maintaining cellular homeostasis. Understanding the link between PRR, autophagy and how its loss results in cell death will be essential for deciphering its role in physiology and pathology.

  11. The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines

    International Nuclear Information System (INIS)

    Autophagy is a process involving the bulk degradation of cellular components in the cytoplasm via the lysosomal degradation pathway. Autophagy manifests a protective role in stressful conditions such as nutrient or growth factor depletion; however, extensive degradation of regulatory molecules or organelles essential for survival can lead to the demise of the cell, or autophagy-mediated cell death. The role of autophagy in cancer is complex with roles in both tumor suppression and tumor promotion proposed. Here we report that an isoform of the C/EBPbeta transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Overexpression of LIP is incompatible with cell growth and when cell cycle analysis was performed, a DNA profile of cells undergoing apoptosis was not observed. Instead, LIP expressing cells appeared to have large autophagic vesicles when examined via electron microscopy. Autophagy was further assessed in LIP expressing cells by monitoring the development of acidic vesicular organelles and conversion of LC3 from the cytoplasmic form to the membrane-bound form. Our work shows that C/EBPbeta isoform, LIP, is another member of the group of transcription factors, including E2F1 and p53, which are capable of playing a role in autophagy.

  12. Detecting Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation.

    Science.gov (United States)

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-01-01

    Autophagy plays an active role during the early stages of embryogenesis in the nematode Caenorhabditis elegans. Although their exact function is unknown, P granules are ribonucleoprotein particles that play a role in germ cell specification. The localization of P granules is restricted to the germline precursor cells in wild-type embryos, as a result of their degradation in the somatic cell lineage. Autophagy is known to be required for the degradation of P granules, as mutations in various autophagy genes, including those encoding the adaptor SEPA-1 and the p62-like adaptor SQST-1, result in the accumulation of the P granule components PGL-1 and PGL-3 (termed PGL granules) in the somatic cells of C. elegans embryos. In this protocol, we present a methodology for using fusion reporters of SEPA-1, SQST-1, and PGL-1 that have aided in the identification of new genes for normal autophagy activity by screening for mutant animals that lack the degradation of these autophagy substrates. PMID:26729906

  13. Increased autophagy and apoptosis contribute to muscle atrophy in a myotonic dystrophy type 1 Drosophila model

    Directory of Open Access Journals (Sweden)

    Ariadna Bargiela

    2015-07-01

    Full Text Available Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1 disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor or muscleblind, or by RNA interference (RNAi-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis.

  14. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Rahul Navale

    Full Text Available Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8 employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64 and aspartic (pepstatin protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine, indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in

  15. Autophagy pathway is required for IL-6 induced neuroendocrine differentiation and chemoresistance of prostate cancer LNCaP cells.

    Directory of Open Access Journals (Sweden)

    Pei-Ching Chang

    Full Text Available Prostate cancer (PCa cells undergoing neuroendocrine differentiation (NED are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA, a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results

  16. Caloric restriction mimetics: natural/physiological pharmacological autophagy inducers.

    Science.gov (United States)

    Mariño, Guillermo; Pietrocola, Federico; Madeo, Frank; Kroemer, Guido

    2014-01-01

    Nutrient depletion, which is one of the physiological triggers of autophagy, results in the depletion of intracellular acetyl coenzyme A (AcCoA) coupled to the deacetylation of cellular proteins. We surmise that there are 3 possibilities to mimic these effects, namely (i) the depletion of cytosolic AcCoA by interfering with its biosynthesis, (ii) the inhibition of acetyltransferases, which are enzymes that transfer acetyl groups from AcCoA to other molecules, mostly leucine residues in cellular proteins, or (iii) the stimulation of deacetylases, which catalyze the removal of acetyl groups from leucine residues. There are several examples of rather nontoxic natural compounds that act as AcCoA depleting agents (e.g., hydroxycitrate), acetyltransferase inhibitors (e.g., anacardic acid, curcumin, epigallocatechin-3-gallate, garcinol, spermidine) or deacetylase activators (e.g., nicotinamide, resveratrol), and that are highly efficient inducers of autophagy in vitro and in vivo, in rodents. Another common characteristic of these agents is their capacity to reduce aging-associated diseases and to confer protective responses against ischemia-induced organ damage. Hence, we classify them as "caloric restriction mimetics" (CRM). Here, we speculate that CRM may mediate their broad health-improving effects by triggering the same molecular pathways that usually are elicited by long-term caloric restriction or short-term starvation and that imply the induction of autophagy as an obligatory event conferring organismal, organ- or cytoprotection. PMID:25484097

  17. Protection and therapy for radiation injuries by expression control of cytokine genes

    International Nuclear Information System (INIS)

    A fibrocyte cell breeding factor, HST-1/FGF-4, a kind of cytokine genes was studied for development of a preventive and treatable medicine for radiation injuries. Expression parts of the factor HST-1/FGF-4 in the intestine of mice were identified using adeno virus vector. The HST-1/FGF-4 decreased an apoptosis in the intestinal cript cells due to irradiation. It was cleared that the breeding factors were important for existence support and repairing process of the intestine by operating on the promotion of wandering and breeding in intestine intraepithelial tissue. The breeding factor appeared in testes of mice, also. The cytokine gene expression, which increased by injuries due to temperature and heat in the testes, indicated a possibility of operating for protection of the tissue injuries. (M. Suetake)

  18. Salmonella enterica serovar Typhimurium lacking hfq gene confers protective immunity against murine typhoid.

    Directory of Open Access Journals (Sweden)

    Uday Shankar Allam

    Full Text Available Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+ T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

  19. Autophagy- An emerging target for melanoma therapy.

    Science.gov (United States)

    Ndoye, Abibatou; Weeraratna, Ashani T

    2016-01-01

    Melanoma accounts for only 5% of all cancers but is the leading cause of skin cancer death due to its high metastatic potential. Patients with metastatic melanoma have a 10-year survival rate of less than 10%. While the clinical landscape for melanoma is evolving rapidly, lack of response to therapies, as well as resistance to therapy remain critical obstacles for treatment of this disease. In recent years, a myriad of therapy resistance mechanisms have been unravelled, one of which is autophagy, the focus of this review. In advanced stages of malignancy, melanoma cells hijack the autophagy machinery in order to alleviate drug-induced and metabolic stress in the tumor microenvironment, thereby promoting resistance to multiple therapies, tumor cell survival, and progression.  Autophagy is an essential cellular process that maintains cellular homeostasis through the recycling of intracellular constituents. Early studies on the role of autophagy in cancer generated controversy as to whether autophagy was pro- or anti-tumorigenic. Currently, there is a consensus that autophagy is tumor-suppressive in the early stages of cancer and tumor-promoting in established tumors.  This review aims to highlight current understandings on the role of autophagy in melanoma malignancy, and specifically therapy resistance; as well as to evaluate recent strategies for therapeutic autophagy modulation. PMID:27583134

  20. Autophagy in allografts rejection: A new direction?

    Science.gov (United States)

    Sun, Hukui; Cheng, Dayan; Ma, Yuanyuan; Wang, Huaiquan; Liang, Ting; Hou, Guihua

    2016-03-18

    Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection is still a major risk for graft survival. Modulating the dosage of immunosuppressive drugs is not a good choice for all patients, new rejection mechanisms discovery are crucial to limit the inflammatory process and preserve the function of the transplant. Autophagy, a fundamental cellular process, can be detected in all subsets of lymphocytes and freshly isolated naive T lymphocytes. It is required for the homeostasis and function of T lymphocytes, which lead to cell survival or cell death depending on the context. T cell receptor (TCR) stimulation and costimulator signals induce strong autophagy, and autophagy deficient T cells leads to rampant apoptosis upon TCR stimulation. Autophagy has been proved to be activated during ischemia-reperfusion (I/R) injury and associated with grafts dysfunction. Furthermore, Autophagy has also emerged as a key mechanism in orchestrating innate and adaptive immune response to self-antigens, which relates with negative selection and Foxp3(+) Treg induction. Although, the role of autophagy in allograft rejection is unknown, current data suggest that autophagy indeed sweeps across both in the graft organs and recipients lymphocytes after transplantation. This review presents the rationale for the hypothesis that targeting the autophagy pathway could be beneficial in promoting graft survival after transplantation. PMID:26876576

  1. Lacritin and other autophagy associated proteins in ocular surface health.

    Science.gov (United States)

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin. PMID:26318608

  2. Role of autophagy in the pathogenesis of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Lee, Jae Keun; Shin, Jin Hee; Lee, Ji Eun; Choi, Eui-Ju

    2015-11-01

    Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease characterized by the selective degeneration of upper and lower motor neurons associated with the abnormal aggregation of ubiquitinated proteins. The molecular mechanisms underlying the pathogenesis of ALS remain unclear, however. Autophagy is a major pathway for the elimination of protein aggregates and damaged organelles and therefore contributes to cellular homeostasis. This catabolic process begins with the formation of the double membrane-bound autophagosome that engulfs portions of the cytoplasm and subsequently fuses with a lysosome to form an autolysosome, in which lysosomal enzymes digest autophagic substrates. Defects at various stages of autophagy have been associated with pathological mutations of several ALS-linked genes including SOD1, p62, TDP-43, and optineurin, suggesting that such defects may play a causative role in the pathogenesis of this condition. In this review, we summarize the dysregulation of autophagy associated with ALS as well as potential therapeutic strategies based on modulation of the autophagic process. PMID:26264610

  3. Mevalonate Pathway Blockade, Mitochondrial Dysfunction and Autophagy: A Possible Link

    Directory of Open Access Journals (Sweden)

    Paola Maura Tricarico

    2015-07-01

    Full Text Available The mevalonate pathway, crucial for cholesterol synthesis, plays a key role in multiple cellular processes. Deregulation of this pathway is also correlated with diminished protein prenylation, an important post-translational modification necessary to localize certain proteins, such as small GTPases, to membranes. Mevalonate pathway blockade has been linked to mitochondrial dysfunction: especially involving lower mitochondrial membrane potential and increased release of pro-apoptotic factors in cytosol. Furthermore a severe reduction of protein prenylation has also been associated with defective autophagy, possibly causing inflammasome activation and subsequent cell death. So, it is tempting to hypothesize a mechanism in which defective autophagy fails to remove damaged mitochondria, resulting in increased cell death. This mechanism could play a significant role in Mevalonate Kinase Deficiency, an autoinflammatory disease characterized by a defect in Mevalonate Kinase, a key enzyme of the mevalonate pathway. Patients carrying mutations in the MVK gene, encoding this enzyme, show increased inflammation and lower protein prenylation levels. This review aims at analysing the correlation between mevalonate pathway defects, mitochondrial dysfunction and defective autophagy, as well as inflammation, using Mevalonate Kinase Deficiency as a model to clarify the current pathogenetic hypothesis as the basis of the disease.

  4. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  5. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hyeon [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  6. Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium.

    Science.gov (United States)

    Perusek, Lindsay; Sahu, Bhubanananda; Parmar, Tanu; Maeno, Hiroshi; Arai, Eisuke; Le, Yun-Zheng; Subauste, Carlos S; Chen, Yu; Palczewski, Krzysztof; Maeda, Akiko

    2015-11-27

    Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7(flox/flox);VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7(flox/flox);VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4(-/-) and Abca4(+/+) backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation. PMID:26468292

  7. Stress management by autophagy: Implications for chemoresistance.

    Science.gov (United States)

    Huang, Zhao; Zhou, Li; Chen, Zhibin; Nice, Edouard C; Huang, Canhua

    2016-07-01

    Development of chemoresistance, which limits the efficiency of anticancer agents, has long been a major problem in cancer therapy and urgently needs to be solved to improve clinical outcomes. Factors contributing to chemoresistance are various, but a key factor is the cell's capability for stress management. Autophagy, a favored survival strategy that organisms employ to get over many kinds of stress, is emerging as a crucial player in drug resistance. It has been shown that autophagy facilitates the resistance of tumor cells to anticancer agents, and abrogation of autophagy could be therapeutically beneficial in some cases, suggesting autophagy could be a promising target for cancer treatments. Thus, defining the roles of autophagy in chemoresistance, and the mechanisms involved, will be critical to enhance the efficiency of chemotherapy and develop novel anticancer strategy interventions. PMID:26757106

  8. Autophagy in the pathogenesis of ankylosing spondylitis.

    Science.gov (United States)

    Ciccia, Francesco; Haroon, Nigil

    2016-06-01

    The pathogenesis of ankylosing spondylitis (AS) is not well understood, and treatment options have met with limited success. Autophagy is a highly conserved mechanism of controlled digestion of damaged organelles within a cell. It helps in the maintenance of cellular homeostasis. The process of autophagy requires the formation of an isolation membrane. They form double-membraned vesicles called "autophagosomes" that engulf a portion of the cytoplasm. Beyond the role in maintenance of cellular homeostasis, autophagy has been demonstrated as one of the most remarkable tools employed by the host cellular defense against bacteria invasion. Autophagy also affects the immune system and thus is implicated in several rheumatic disease processes. In this article, we explore the potential role of autophagy in the pathogenesis of AS. PMID:27075464

  9. Autophagy and the immune function in aging.

    Science.gov (United States)

    Cuervo, Ana Maria; Macian, Fernando

    2014-08-01

    Just when you thought that you had heard it all about autophagy-the conserved cellular process that mediates turnover of cellular constituents in the lysosomes - studies keep coming out highlighting new types of autophagy, new functions for autophagy or even new autophagy-independent roles for the proteins associated with this process. The field of immunology has been riding the autophagic wave since the beginning of its revival; first due to its role in the host defense against pathogens, and more recently through the better understanding of the unique characteristics and functions of different autophagic pathways in immune cells. Here, we describe some of these new functions that are tightening the connection between autophagy and acquired or innate immunity and their malfunctioning with age. PMID:24929664

  10. Autophagy, inflammation and innate immunity in inflammatory myopathies.

    Directory of Open Access Journals (Sweden)

    Cristina Cappelletti

    Full Text Available Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs. In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM, polymyositis (PM, dermatomyositis (DM and juvenile dermatomyositis (JDM. We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1. These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous 'danger signal'. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types.

  11. The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum

    Science.gov (United States)

    Cervantes, Serena; Bunnik, Evelien M; Saraf, Anita; Conner, Christopher M; Escalante, Aster; Sardiu, Mihaela E; Ponts, Nadia; Prudhomme, Jacques; Florens, Laurence; Le Roch, Karine G

    2014-01-01

    Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies. PMID:24275162

  12. Inhibition of mTOR-dependent autophagy sensitizes leukemic cells to cytarabine-induced apoptotic death.

    Directory of Open Access Journals (Sweden)

    Mihajlo Bosnjak

    Full Text Available The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4 and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR, and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.

  13. Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis.

    Science.gov (United States)

    Lee, Hae-Youn; Kim, Jinyoung; Quan, Wenying; Lee, June-Chul; Kim, Min-Soo; Kim, Seok-Hyung; Bae, Jin-Woo; Hur, Kyu Yeon; Lee, Myung-Shik

    2016-08-01

    Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD(+):NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis. PMID:27337687

  14. CDDO-Im protects from acetaminophen hepatotoxicity through induction of Nrf2-dependent genes

    International Nuclear Information System (INIS)

    CDDO-Im is a synthetic triterpenoid recently shown to induce cytoprotective genes through the Nrf2-Keap1 pathway, an important mechanism for the induction of cytoprotective genes in response to oxidative stress. Upon oxidative or electrophilic insult, the transcription factor Nrf2 translocates to the nucleus, heterodimerizes with small Maf proteins, and binds to antioxidant response elements (AREs) in the upstream promoter regions of various cytoprotective genes. To further elucidate the hepatoprotective effects of CDDO-Im, wild-type and Nrf2-null mice were pretreated with CDDO-Im (1 mg/kg, i.p.) or vehicle (DMSO), and then administered acetaminophen (500 mg/kg, i.p.). Pretreatment of wild-type mice with CDDO-Im reduced liver injury caused by acetaminophen. In contrast, hepatoprotection by CDDO-Im was not observed in Nrf2-null mice. CDDO-Im increased Nrf2 protein expression and Nrf2-ARE binding in wild-type, but not Nrf2-null mice. Furthermore, CDDO-Im increased the mRNA expression of the Nrf2 target genes NAD(P)H: quinone oxidoreductase-1 (Nqo1); glutamate-cysteine ligase, catalytic subunit (Gclc); and heme-oxygenase-1 (Ho-1), in both a dose- and time-dependent manner. Conversely, CDDO-Im did not induce Nqo1, Gclc, and Ho-1 mRNA expression in Nrf2-null mice. Collectively, the present study shows that CDDO-Im pretreatment induces Nrf2-dependent cytoprotective genes and protects the liver from acetaminophen-induced hepatic injury

  15. Autophagy and bacterial clearance: a not so clear picture

    OpenAIRE

    Mostowy, Serge

    2012-01-01

    Autophagy, an intracellular degradation process highly conserved from yeast to humans, is viewed as an important defence mechanism to clear intracellular bacteria. However, recent work has shown that autophagy may have different roles during different bacterial infections that restrict bacterial replication (antibacterial autophagy), act in cell autonomous signalling (non-bacterial autophagy) or support bacterial replication (pro-bacterial autophagy). This review will focus on newfound intera...

  16. A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy

    NARCIS (Netherlands)

    Stasyk, OV; van der Klei, IJ; Bellu, AR; Shen, SG; Kiel, JAKW; Cregg, JM; Veenhuis, M; Stasyk, Oleh V.; Shen, Shigang; Cregg, James M.

    1999-01-01

    Methylotrophic yeasts contain large peroxisomes during growth on methanol. Upon exposure to excess glucose or ethanol these organelles are selectively degraded by autophagy, Here we describe the cloning of a Pichia pastoris gene (PpVPS15) involved ill peroxisome degradation, which is homologous to S

  17. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis.

    Science.gov (United States)

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M

    2008-09-22

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53-as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo. PMID:18794330

  18. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization.

    Science.gov (United States)

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  19. THE RISK OF GENE TRANSFERRING IN THE INSURANCE PROTECTION OF AGRICULTERE

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-05-01

    Full Text Available The paper justified essence of genetic engineering as the object of insurance services. Defines the concept of risk gene transferring. The character features of this specific risk. The influence and consequences for agricultural producers. The description of the possible creation of the concept of insurance services that cover risk of gene transferring. The study reveals of the use of GMOs in agriculture, due to issues of economic security of a particular region or country as a whole. To determined the impact of risks and control for developing and developed countries that are important aspects of farming. Changes in weather, climate, productivity, price values, public policy, the situation on global markets can cause large fluctuations in agricultural production, and consequently affecting the income of agricultural producers. Risk management includes a range of strategies that reduce the social and financial implications of possible changes affecting the production and income of farmers. There is a need for an in-depth study of the theoretical and practical aspects of the impact of the risk of gene transferring in the context of insurance protection.

  20. Beclin1-induced autophagy abrogates radioresistance of lung cancer cells by suppressing osteopontin

    International Nuclear Information System (INIS)

    Osteopontin (OPN) serves as an indicator of resistance to radiotherapy. However, the role of OPN in the development of acquired radioresistance in human lung cancer cells has not yet been fully elucidated. Therefore, the potential importance of OPN as a marker of lung cancer with a potential significant role in the development of radioresistance against repeated radiotherapy has prompted us to define the pathways by which OPN regulates lung cancer cell growth. In addition, autophagy has been reported to play a key role in the radiosensitization of cancer cells. Here, we report that increased OPN expression through induction of nuclear p53 following irradiation was inhibited by exogenous beclin-1 (BECN1). Our results clearly show that BECN1 gene expression led to induction of autophagy and inhibition of cancer cell growth and angiogenesis. Our results suggest that the induction of autophagy abrogated the radioresistance of the cancer cells. Interestingly, we showed that knockdown of OPN by lentivirus-mediated shRNA induced the autophagy of human lung cancer cell. Taken together, these results suggest that OPN and BECN1 can be molecular targets for overcoming radioresistance by controlling autophagy. (author)

  1. Autophagy may occur at an early stage of cholangiocarcinogenesis via biliary intraepithelial neoplasia.

    Science.gov (United States)

    Sasaki, Motoko; Nitta, Takeo; Sato, Yasunori; Nakanuma, Yasuni

    2015-02-01

    Similar to the pancreatic carcinoma sequence model, cholangiocarcinoma reportedly follows a stepwise carcinogenesis process via the precursor lesion biliary intraepithelial neoplasia (BilIN). Given that autophagy plays an important role in the occurrence and development of carcinomas, we examined the involvement of autophagy in multistep cholangiocarcinogenesis. Thirty-six patients with hepatolithiasis associated with BilIN and/or cholangiocarcinoma, 7 with intrahepatic cholangiocarcinoma, 8 with intraductal papillary neoplasm of the bile duct (IPNB), and 6 with control livers were surveyed. Their lesions were categorized as follows: invasive carcinoma (n = 16), IPNB (n = 8), BIlN-3 (n = 16), BilIN-1/2 (n = 40), nonneoplastic large bile duct (n = 55), and peribiliary gland (n = 55). We examined the immunohistochemical expression of autophagy-related proteins, microtubule-associated proteins light chain 3β (LC3), beclin-1, and p62/sequestosome-1 (p62), as well as tumor suppressor gene product p53. The extent of expression was semiquantitatively assessed. The status of KRAS mutations at codons 12 and 13 was examined in selected cases of BilIN-1/2. The expression of LC3 (cytoplasmic), beclin-1 (cytoplasmic), and p62 (cytoplasmic and nuclear) was significantly higher in BilIN-1/2, BilIN-3, IPNB, and invasive carcinoma than in large bile duct and peribiliary gland (P cholangiocarcinogenesis in hepatolithiasis. Autophagy, probably deregulated autophagy, may be related to the occurrence and development of cholangiocarcinoma. PMID:25466963

  2. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    Science.gov (United States)

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  3. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  4. Induction of Autophagy in the Striatum and Hypothalamus of Mice after 835 MHz Radiofrequency Exposure.

    Science.gov (United States)

    Kim, Ju Hwan; Huh, Yang Hoon; Kim, Hak Rim

    2016-01-01

    The extensive use of wireless mobile phones and associated communication devices has led to increasing public concern about potential biological health-related effects of the exposure to electromagnetic fields (EMFs). EMFs emitted by a mobile phone have been suggested to influence neuronal functions in the brain and affect behavior. However, the affects and phenotype of EMFs exposure are unclear. We applied radiofrequency (RF) of 835 MHz at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day for 4 and 12 weeks to clarify the biological effects on mouse brain. Interestingly, microarray data indicated that a variety of autophagic related genes showed fold-change within small range after 835 MHz RF exposure. qRT-PCR revealed significant up-regulation of the autophagic genes Atg5, LC3A and LC3B in the striatum and hypothalamus after a 12-week RF. In parallel, protein expression of LC3B-II was also increased in both brain regions. Autophagosomes were observed in the striatum and hypothalamus of RF-exposed mice, based on neuronal transmission electron microscopy. Taken together, the results indicate that RF exposure of the brain can induce autophagy in neuronal tissues, providing insight into the protective mechanism or adaptation to RF stress. PMID:27073885

  5. Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Zhongxia Wang

    2014-01-01

    Full Text Available Background. Hepatocellular carcinoma (HCC remains a disastrous disease and the treatment for HCC is rather limited. Separation and identification of active compounds from traditionally used herbs in HCC treatment may shed light on novel therapeutic drugs for HCC. Methods. Cell viability and colony forming assay were conducted to determine anti-HCC activity. Morphology of cells and activity of caspases were analyzed. Antiapoptotic Bcl-2 family proteins and JNK were also examined. Levels of unfolded protein response (UPR markers were determined and intracellular calcium was assayed. Small interfering RNAs (siRNAs were used to investigate the role of UPR and autophagy in baicalein-induced cell death. Results. Among four studied flavonoids, only baicalein exhibited satisfactory inhibition of viability and colony formation of HCC cells within water-soluble concentration. Baicalein induced apoptosis via endoplasmic reticulum (ER stress, possibly by downregulating prosurvival Bcl-2 family, increasing intracellular calcium, and activating JNK. CHOP was the executor of cell death during baicalein-induced ER stress while eIF2α and IRE1α played protective roles. Protective autophagy was also triggered by baicalein in HCC cells. Conclusion. Baicalein exhibits prominent anti-HCC activity. This flavonoid induces apoptosis and protective autophagy via ER stress. Combination of baicalein and autophagy inhibitors may represent a promising therapy against HCC.

  6. Role of fractionated ionizing radiation in autophagy in drug resistance of ovarian cancer

    International Nuclear Information System (INIS)

    Objective: To detect the effects of different subtypes of fractionated ionizing radiation (IR) on autophagy in ovarian cancer cell line and to explore the relevant mechanisms. Methods: The cells were divided into control group, fractionated IR group (2 Gy ·d-1×5) and single IR group (10 Gy ·d-1×1). MTT assay was used to detect the drug sensitivities of the cells in three groups to VCR, VP-16 and DDP. MDC staining was used to examine the incidence of autophagy, Real-time fluorescence quantitative PCR was used to detect the expression levels of autophagy-relative genes MAPLC3B and Akt1 mRNA. Western blotting was applied to detect the expression of autophagy-related protein MAPLC3B, protein kinase B (PKB, Akt1)/mammalian target of rapamycin (mTOR) and its downstream gene P70 S6K. Results: Compared with control group, IR increased the drug sensitivities of SKVCR cells to VCR and VP-16, which was more significant in fractionated group (P<0.05). Compared with control group, IR increased the incidence of autophagy, particularly in fractionated IR group. Compared with control group, MAPLC3B was increased and Akt1 was decreased (P<0.05) after irradiation both at mRNA level and protein level, the expressions of mTOR, p-mTOR, P70 S6K, p-P70 S6K were decreased, the changes in fractionated IR group were more significant compared with single IR group. Conclusion: Different subtypes of IR can induce the cell death of ovarian cancer, the Akt1/mTOR/S6K signal pathway autophagy might be involved in the regulation mechanism. (authors)

  7. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    International Nuclear Information System (INIS)

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  8. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  9. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    Directory of Open Access Journals (Sweden)

    Chen Dishi

    2011-06-01

    Full Text Available Abstract Background Porcine parvovirus (PPV VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs with similar morphology to the native capsid. Here, a pseudorabies virus (PRV system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28 following virulent PPV challenge compared with the control (7 of 31. Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  10. RAP1 Protects from Obesity through Its Extratelomeric Role Regulating Gene Expression

    Directory of Open Access Journals (Sweden)

    Paula Martínez

    2013-06-01

    Full Text Available RAP1 is part of shelterin, the protective complex at telomeres. RAP1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of RAP1 in vivo, we generated a RAP1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARα signaling. We identify Pparα and Pgc1α as key factors affected by Rap1 deletion in the liver. We show that RAP1 binds to Pparα and Pgc1α loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism.

  11. Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

    OpenAIRE

    Motoki, Atsuko; Merkel, Matthias J.; Packwood, William H.; Cao, Zhiping; Liu, Lijuan; Iliff, Jeffrey; Alkayed, Nabil J.; Van Winkle, Donna M.

    2008-01-01

    Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ...

  12. Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly.

    Science.gov (United States)

    Lapaquette, Pierre; Glasser, Anne-Lise; Huett, Alan; Xavier, Ramnik J; Darfeuille-Michaud, Arlette

    2010-01-01

    Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation. PMID:19747213

  13. Hypercapnia Inhibits Autophagy and Bacterial Killing in Human Macrophages by Increasing Expression of Bcl-2 and Bcl-xL

    Science.gov (United States)

    Casalino-Matsuda, S. Marina; Nair, Aisha; Beitel, Greg J.; Gates, Khalilah L.; Sporn, Peter H. S.

    2015-01-01

    Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in patients with advanced lung disease and severe pulmonary infections, and is associated with high mortality. We previously reported that hypercapnia alters expression of host defense genes, inhibits phagocytosis, and increases the mortality of Pseudomonas pneumonia in mice. However, the effect of hypercapnia on autophagy, a conserved process by which cells sequester and degrade proteins and damaged organelles that also plays a key role in antimicrobial host defense and pathogen clearance, has not previously been examined. In the present study we show that hypercapnia inhibits autophagy induced by starvation, rapamycin, LPS, heat-killed and live bacteria in the human macrophage. Inhibition of autophagy by elevated CO2 was not attributable to acidosis. Hypercapnia also reduced macrophage killing of Pseudomonas aeruginosa. Moreover, elevated CO2 induced the expression of Bcl-2 and Bcl-xL, anti-apoptotic factors that negatively regulate autophagy by blocking Beclin 1, an essential component of the autophagy initiation complex. Furthermore, siRNA targeting Bcl-2 and Bcl-xL and the small molecule Z36, which blocks Bcl-2 and Bcl-xL binding to Beclin 1, prevented hypercapnic inhibition of autophagy and bacterial killing. These results suggest that targeting the Bcl-2/Bcl-xL-Beclin 1 interaction may hold promise for ameliorating hypercapnia-induced immunosuppression and improving resistance to infection in patients with advanced lung disease and hypercapnia. PMID:25895534

  14. The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.

    OpenAIRE

    Lin, S J; Culotta, V C

    1995-01-01

    In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase. Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species. This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD. ATX1 encodes a 8.2-kDa poly...

  15. Effect of P39 Gene Deletion in Live Brucella Vaccine Strains on Residual Virulence and Protective Activity in Mice

    OpenAIRE

    Tibor, Anne; Jacques, Isabelle; Guilloteau, Laurence; Verger, Jean-Michel; Grayon, Maggy; Wansard, Valerie; Letesson, Jean-Jacques

    1998-01-01

    The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could pr...

  16. Autophagy in lung disease pathogenesis and therapeutics

    OpenAIRE

    Ryter, Stefan W.; Augustine M K Choi

    2015-01-01

    Autophagy, a cellular pathway for the degradation of damaged organelles and proteins, has gained increasing importance in human pulmonary diseases, both as a modulator of pathogenesis and as a potential therapeutic target. In this pathway, cytosolic cargos are sequestered into autophagosomes, which are delivered to the lysosomes where they are enzymatically degraded and then recycled as metabolic precursors. Autophagy exerts an important effector function in the regulation of inflammation, an...

  17. Skeletal Muscle Autophagy: A New Metabolic Regulator

    OpenAIRE

    Neel, Brian A.; Lin, Yuxi; Pessin, Jeffrey E.

    2013-01-01

    Autophagy classically functions as a physiological process to degrade cytoplasmic components, protein aggregates, and/or organelles, as a mechanism for nutrient breakdown, and as a regulator of cellular architecture. Proper autophagic flux is vital for both functional skeletal muscle, which controls support and movement of the skeleton, and muscle metabolism. The role of autophagy as a metabolic regulator in muscle has been previously studied; however, the underlying molecular mechanisms that...

  18. The role of autophagy in Parkinson's disease☆

    OpenAIRE

    Zhang, Lei; Dong, Yaru; Xu, Xiaoheng; Xu, Zhong

    2012-01-01

    Although Parkinson's disease is the most common neurodegenerative movement disorder, the mechanisms of pathogenesis remain poorly understood. Recent findings have shown that deregulation of the autophagy-lysosome pathway is involved in the pathogenesis of Parkinson's disease. This review summarizes the most recent findings and discusses the unique role of the autophagy-lysosome pathway in Parkinson's disease to highlight the possibility of Parkinson's disease treatment strategies that incorpo...

  19. Mechanisms of mitochondria and autophagy crosstalk

    OpenAIRE

    Rambold, Angelika S.; Lippincott-Schwartz, Jennifer

    2011-01-01

    Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuro...

  20. Rapamycin requires AMPK activity and p27 expression for promoting autophagy-dependent Tsc2-null cell survival.

    Science.gov (United States)

    Campos, Tania; Ziehe, Javiera; Fuentes-Villalobos, Francisco; Riquelme, Orlando; Peña, Daniela; Troncoso, Rodrigo; Lavandero, Sergio; Morin, Violeta; Pincheira, Roxana; Castro, Ariel F

    2016-06-01

    Tuberous sclerosis complex (TSC) disease results from inactivation of the TSC1 or TSC2 gene, and is characterized by benign tumors in several organs. Because TSC tumorigenesis correlates with hyperactivation of mTORC1, current therapies focus on mTORC1 inhibition with rapamycin or its analogs. Rapamycin-induced tumor shrinkage has been reported, but tumor recurrence occurs on withdrawal from rapamycin. Autophagy has been associated with development of TSC tumors and with tumor cell survival during rapamycin treatment. mTORC1 and AMPK directly inhibit and activate autophagy, respectively. AMPK is hyperactivated in TSC cells and tumors, and drives cytoplasmic sequestration of the cell-cycle inhibitor p27KIP (p27). Whether AMPK and p27 are involved in rapamycin-induced autophagy and survival of TSC cells remain unexplored. Here, we show that inhibition of AMPK by compound C or by shRNA-mediated depletion of LKB1 reduces activation of autophagy by rapamycin in Tsc2-null cells. Similarly, shRNA-mediated depletion of p27 inhibited rapamycin-induced autophagy. In support of p27 lying downstream of AMPK on the activation of autophagy in Tsc2-null cells, a p27 mutant that preferentially localizes in the cytosol recovered the effect of rapamycin on autophagy in both p27- and LKB1-depleted cells, but a nuclear p27 mutant was inactive. Finally, we show that p27-dependent activation of autophagy is involved in Tsc2-null cell survival under rapamycin treatment. These results indicate that an AMPK/p27 axis is promoting a survival mechanism that could explain in part the relapse of TSC tumors treated with rapamycin, exposing new avenues for designing more efficient treatments for TSC patients. PMID:26975583

  1. Autophagy inhibition plays the synergetic killing roles with radiation in the multi-drug resistant SKVCR ovarian cancer cells

    International Nuclear Information System (INIS)

    Autophagy has attracted attentions as a novel mechanism for tumor development. In this study Human ovarian carcinoma cell line SKOV3 and multidrug-resistant phenotype SKVCR cells were used and the roles of autophagy in radiation-induced cell death were analyzed. Cell viability was examined by colony formation and cell counting kit-8 (CCK-8) assay, 3MA and ZVAD were used to block autophagy and apoptosis, respectively. Quantitative real-time PCR was used to detect mRNA level and Western blot was used to detect protein expression, monodansylcadaverine (MDC) staining and flow cytometery were used for autophagy, apoptosis and cell cycle dynamics, respectively. (1) The radiosensitivity exhibited differently in SKOV3 and SKVCR cells (SKOV3: D0=3.37, SKVCR: D0= 4.18); compared with SKOV3 the constitutive expression of MAPLC3 in SKVCR was higher, but no change of Caspase-3 and cleaved Caspase-3. (2) The ionizing radiation (IR)- induced apoptosis and autophagy were significant in both cells (P<0.05); inhibition of apoptosis with ZVAD showed no impact on survival of SKOV3 and SKVCR cells after radiation, while inhibition of autophagy significantly decreased viability in SKVCR cells, for SKVO3 cells only low level of radiation (2 Gy and 4 Gy) could decrease the viability(P<0.05). (3) ZVAD inhibited apoptosis and autophagy in both cells, 3MA inhibit apoptosis in SKOV3, and promote apoptosis in SKVCR, together with inhibition of autophagy. (4) G2/M arrest was induced by radiation in both cells; the accumulation of G2/M was more significant in SKOV3, 3MA attenuated the radiation-induced S phase delay in SKVCR. IR-induced autophagy provides a self-protective mechanism against radiotherapy in SKVCR cells, the use of autophagy inhibitor, 3MA, increases the killing effects of radiation by inhibiting autophagy and radiation- induced S phase delay, also by the increase of apoptosis, which suggests a better therapeutic strategy in drug- resistant SKVCR ovarian cancer cells

  2. Autophagy in the control of food intake.

    Science.gov (United States)

    Singh, Rajat

    2012-04-01

    The cellular nutrient sensing apparatus detects nutritional depletion and transmits this information to downstream effectors that generate energy from alternate sources. Autophagy is a crucial catabolic pathway that turns over redundant cytoplasmic components in lysosomes to provide energy to the starved cell. Recent studies have described a role for hypothalamic autophagy in the control of food intake and energy balance. Activated autophagy in hypothalamic neurons during starvation mobilized neuron-intrinsic lipids to generate free fatty acids that increased AgRP levels. AgRP neuron-specific inhibition of autophagy decreased fasting-induced increases in AgRP levels and food intake. Deletion of autophagy in AgRP neurons led to constitutive increases in levels of proopiomelanocortin and its active processed product, α-melanocyte stimulating hormone that contributed to reduced adiposity in these rodents. The current manuscript discusses these new findings and raises additional questions that may help understand how hypothalamic autophagy controls food intake and energy balance. These studies may have implications for designing new therapies against obesity and insulin resistance. PMID:23700515

  3. Absolute Protection and Relative Protection:Comparative Thinking of Gene Patent Protection Principle%绝对保护与相对保护:基因专利保护原则的选择

    Institute of Scientific and Technical Information of China (English)

    高莉

    2014-01-01

    Although most countries agree,to a certain extent,that gene has patent qualification,issues such as the scope of protection of gene patent,patent assessment are still the focus of the argument in recent years. Absolute protection and relative protection are now mainly taken by all countries on the patent protection of gene two principles,which define the scope of protection of gene patent. Each has its own advantages and disadvantages. No matter what kind of protection prin-ciple is used alone,it is biased. At the same time,although the relative protectionism,also known as "functional limit"principle is proposed based on the criticism of the absolute protection,its adoption of patent qualification has caused new influence and brought new challenges.%尽管大多数国家都在一定程度上承认基因具有可专利性资格,然而,基因专利保护范围的界定、可专利性审查等问题仍然是近年来争论的焦点。绝对保护与相对保护是目前各国主要采用的有关基因专利保护的两种原则,这两种原则对于基因专利保护范围的界定各有利弊,如单独适用会有失偏颇。同时,虽然相对保护主义,是在对绝对保护主义批判的基础上提出的,但是它的采用可能影响可专利性资格的确定。因此,两种原则应因情况,融合适用。

  4. Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum

    DEFF Research Database (Denmark)

    Josefsen, Lone; Droce, Aida; Søndergaard, Teis;

    2012-01-01

    The role of autophagy in necrotrophic fungal physiology and infection biology is poorly understood. We have studied autophagy in the necrotrophic plant pathogen Fusarium graminearum in relation to development of nonassimilating structures and infection. We identified an ATG8 homolog F. graminearum...... ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in Delta Fgatg8 fully restores the WT phenotype. The results showed that autophagy plays a...... pivotal role for supplying nutrients to nonassimilating structures necessary for growth and is important for plant colonization. This also indicates that autophagy is a central mechanism for fungal adaptation to nonoptimal C/N ratios....

  5. The cooperation between the autophagy machinery and the inflammasome to implement an appropriate innate immune response: do they regulate each other?

    OpenAIRE

    Abdelaziz, Dalia H. A.; Khalil, Hany; Cormet-Boyaka, Estelle; Amer, Amal O.

    2015-01-01

    Autophagy is originally described as the main catabolic pathway responsible for maintaining intracellular nutritional homeosta-sis that involves the formation of a unique vacuole, the autophago-some, and the interaction with the endosome-lysosome pathways. This conserved machinery plays a key role in immune-protection against different invaders, including pathogenic bacteria, intracellular parasites, and some viruses like herpes simplex and hepatitis C virus. Importantly, autophagy is linked ...

  6. Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling.

    Directory of Open Access Journals (Sweden)

    Dong-Min Shin

    Full Text Available The "enhanced intracellular survival" (eis gene of Mycobacterium tuberculosis (Mtb is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

  7. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

    Science.gov (United States)

    Ejaz, Asim; Mitterberger, Maria C; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C; Zwerschke, Werner

    2016-04-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells. PMID:27211557

  8. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt–mTOR Inhibition

    Science.gov (United States)

    Ejaz, Asim; Mitterberger, Maria C.; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E.; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C.; Zwerschke, Werner

    2016-01-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1–DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt–mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells.

  9. Characterization of early autophagy signaling by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer Tg; Zarei, Mostafa; Sprenger, Adrian;

    2014-01-01

    Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flu...

  10. Autophagy as a target for cancer therapy: new developments

    International Nuclear Information System (INIS)

    Autophagy is an evolutionarily conserved lysosomal degradation pathway that eliminates cytosolic proteins, macromolecules, organelles, and protein aggregates. Activation of autophagy may function as a tumor suppressor by degrading defective organelles and other cellular components. However, this pathway may also be exploited by cancer cells to generate nutrients and energy during periods of starvation, hypoxia, and stress induced by chemotherapy. Therefore, induction of autophagy has emerged as a drug resistance mechanism that promotes cancer cell survival via self-digestion. Numerous preclinical studies have demonstrated that inhibition of autophagy enhances the activity of a broad array of anticancer agents. Thus, targeting autophagy may be a global anticancer strategy that may improve the efficacy of many standard of care agents. These results have led to multiple clinical trials to evaluate autophagy inhibition in combination with conventional chemotherapy. In this review, we summarize the anticancer agents that have been reported to modulate autophagy and discuss new developments in autophagy inhibition as an anticancer strategy

  11. Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

    International Nuclear Information System (INIS)

    ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel. Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts. ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest. ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest

  12. Re-expression of ARHI (DIRAS3 induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

    Directory of Open Access Journals (Sweden)

    Bast Robert C

    2011-01-01

    Full Text Available Abstract Background ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel. Methods Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts. Results ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest. Conclusions ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.

  13. 6-Gingerol Protects against Nutritional Steatohepatitis by Regulating Key Genes Related to Inflammation and Lipid Metabolism

    Directory of Open Access Journals (Sweden)

    Thing-Fong Tzeng

    2015-02-01

    Full Text Available Non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH, appears to be increasingly common worldwide. The aim of the study was to investigate the effects of 6-gingerol ((S-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl-3-decanone, a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of NASH. In HepG2 cells, 6-gingerol (100 μmol/L treatment inhibited free fatty acids mixture (0.33 mmol/L palmitate and 0.66 mmol/L oleate-induced triglyceride and inflammatory marker accumulations. Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD diet to induce steatohepatitis. After four weeks of MCD diet feeding, the mice were dosed orally with 6-gingerol (25, 50 or 100 mg/kg/day once daily for another four weeks. 6-Gingerol (100 mg/kg/day attenuated liver steatosis and necro-inflammation in MCD diet-fed mice. The expressions of inflammatory cytokine genes, including those for monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and nuclear transcription factor (NF-κB, which were increased in the livers of MCD diet-fed mice, were attenuated by 6-gingerol. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with induction of peroxisome proliferator-activated receptor α. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to lipid metabolism and inflammation.

  14. Centralized Consensus Hemagglutinin Genes Induce Protective Immunity against H1, H3 and H5 Influenza Viruses.

    Science.gov (United States)

    Webby, Richard J; Weaver, Eric A

    2015-01-01

    With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940's. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA) genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 1010, 5 X 107 and 1 X 107 virus particles per mouse to represent high, intermediate and low doses, respectively. 100% of the mice that were vaccinated with the high dose vaccine were protected from heterologous lethal challenges within each subtype. In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines. PMID:26469190

  15. Dual role of autophagy in HIV-1 replication and pathogenesis

    OpenAIRE

    Killian M

    2012-01-01

    Abstract Autophagy, the major mechanism for degrading long-lived intracellular proteins and organelles, is essential for eukaryotic cell homeostasis. Autophagy also defends the cell against invasion by microorganisms and has important roles in innate and adaptive immunity. Increasingly evident is that HIV-1 replication is dependent on select components of autophagy. Fittingly, HIV-1 proteins are able to modulate autophagy to maximize virus production. At the same time, HIV-1 proteins appear t...

  16. Bone Cell Autophagy Is Regulated by Environmental Factors

    OpenAIRE

    Zahm, Adam M.; Bohensky, Jolene; Adams, Christopher S.; Shapiro, Irving M.; Srinivas, Vickram

    2011-01-01

    The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient d...

  17. Interactions between Shigella flexneri and the Autophagy Machinery

    OpenAIRE

    Krokowski, Sina; Mostowy, Serge

    2016-01-01

    Autophagy, an intracellular degradation process, is increasingly recognized as having important roles in host defense. Interactions between Shigella flexneri and the autophagy machinery were first discovered in 2005. Since then, work has shown that multiple autophagy pathways are triggered by S. flexneri, and autophagic responses can have different roles during Shigella infection. Here, we review the interactions between S. flexneri and the autophagy machinery, highlighting that studies using...

  18. ZINC-mediated gene expression offers protection against H2O2-induced cytotoxicity

    International Nuclear Information System (INIS)

    The ability of zinc to mobilize defense against reactive oxygen species (ROS) and H2O2-induced apoptosis was studied using a primary culture of rainbow trout gill cells. Gill cells were pretreated for 24 h with 100 μM ZnSO4 followed by 24-h exposure to 100 or 200 μM H2O2, or were subjected to 100 μM ZnSO4 together with 100 or 200 μM H2O2. Metallothionein-A (MTA) and metallothionein-B (MTB) mRNA levels were increased after treatment with zinc or H2O2, separately or in combination. Similarly, mRNA for glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD) were increased in response to either zinc or H2O2, or after sequential treatments with zinc followed by H2O2. The stimulatory effects of zinc or H2O2 on MTA, MTB, GST, and G6PD mRNA levels could be blocked by addition of the membrane permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suggesting that H2O2-induced upregulation of these genes is zinc-dependent. Pretreatment with zinc protected the cells from subsequent cell damage and apoptosis, as assessed by lactate dehydrogenase leakage, mitochondrial dehydrogenase activity (MTT assay), caspase-3 activity, and DNA fragmentation. In contrast, when gill cells were coincubated with zinc and H2O2 at the same time, H2O2 toxicity was higher than after treatment with H2O2 alone. It is concluded that zinc had a direct pro-oxidant effect when administered together with H2O2, but that pretreatment of zinc inhibited cytotoxicity and apoptosis through an indirect antioxidant action. We propose that the antioxidant action is manifested through zinc-dependent expression of several genes encoding antioxidant proteins (e.g., MTA, MTB, G6PD, and GST). Furthermore, the apparent zinc-dependency of H2O2-induced expression of antioxidant genes suggests that zinc might act as a physiological signal to mediate the response to oxidative stress

  19. Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell

    International Nuclear Information System (INIS)

    Highlights: ► miR-199a-5p levels were significantly decreased after cisplatin treatment. ► Cisplatin treatment induced autophagy activation. ► Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell. -- Abstract: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Systemic chemotherapy plays an important role in the treatment of patients with advanced liver cancer. However, chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic, and the underlying mechanism of such resistance is not fully understood. In the study, we found that miR-199a-5p levels were significantly reduced in HCC patients treated with cisplatin-based chemotherapy. Cisplatin treatment also resulted in decreased miR-199a-5p levels in human HCC cell lines. Forced expression of miR-199a-5p promoted cisplatin-induced inhibition of cell proliferation. Cisplatin treatment activated autophagy in Huh7 and HepG2 cells, which increased cell proliferation. We further demonstrated that downregulated miR-199a-5p enhanced autophagy activation by targeting autophagy-associated gene 7 (ATG7). More important, autophagy inhibition abrogated miR-199a-5p downregulation-induced cell proliferation. These data demonstrated that miR-199a-5p/autophagy signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.

  20. Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model.

    Science.gov (United States)

    Vaisburd, Sinaya; Shemer, Zeev; Yeheskel, Adva; Giladi, Eliezer; Gozes, Illana

    2015-01-01

    Mutated disrupted in schizophrenia 1 (DISC1), a microtubule regulating protein, leads to schizophrenia and other psychiatric illnesses. It is hypothesized that microtubule stabilization may provide neuroprotection in schizophrenia. The NAP (NAPVSIPQ) sequence of activity-dependent neuroprotective protein (ADNP) contains the SxIP motif, microtubule end binding (EB) protein target, which is critical for microtubule dynamics leading to synaptic plasticity and neuroprotection. Bioinformatics prediction for FDA approved drugs mimicking SxIP-like motif which displace NAP-EB binding identified Risperidone. Risperidone or NAP effectively ameliorated object recognition deficits in the mutated DISC1 mouse model. NAP but not Risperidone, reduced anxiety in the mutated mice. Doxycycline, which blocked the expression of the mutated DISC1, did not reverse the phenotype. Transcripts of Forkhead-BOX P2 (Foxp2), a gene regulating DISC1 and associated with human ability to acquire a spoken language, were increased in the hippocampus of the DISC1 mutated mice and were significantly lowered after treatment with NAP, Risperidone, or the combination of both. Thus, the combination of NAP and standard of care Risperidone in humans may protect against language disturbances associated with negative and cognitive impairments in schizophrenia. PMID:26553741

  1. Protective and Susceptible HLA Class I Genes in Patients with End-Stage Renal Disease

    Directory of Open Access Journals (Sweden)

    2008-01-01

    Full Text Available The role of the HLA system in the pathophysiology of primary renal disease is intriguing, but not completely resolved. According to the results of studies links between HLA haplotype and renal failure has been reported. This study was conducted to determine protective and susceptible role of HLA class I genes in end stage renal disease patients. Subjects of this study were 77 individuals from Azerbaijan republic referred to Iran Red Crescent Society clinic in Baku of which were assigned into 2 group, case and control, based on renal disease. Case group were 26 patients with end stage renal disease candidate for renal transplant and controls were 51 healthy subjects. Typing of HLA class I was performed by serologic method. There was no significant difference in age and sex between control and patient groups. The most frequent detected HLA antigens were A2 (41.6%, A3(28.6%, A24(26% from A loci and B35 (46.8%, B51 (29.9%, B18 (13% from B loci. Significant association was found between susceptibility to ESRD and HLA-A33, A11, B49 (p<0.05. The findings support the idea that polymorphism of HLA class I may influence the susceptibility to ESRD. We suggested HLA antigen distribution will identify the high-risk patients who are candidates for transplantation.

  2. Autophagy and Retromer Components in Plant Innate Immunity

    DEFF Research Database (Denmark)

    Munch, David

    -hormone salicylic acid. Here, I present data that make it clear that NPR1 does not directly regulate autophagy, but instead control stress responses that indirectly activate autophagy. The observations presented will also clarify why autophagy has been described as being both a pro-death and pro-life pathway under...

  3. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Aminov, R I; Garrigues-Jeanjean, N; Mackie, R I

    2001-01-01

    Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals. PMID:11133424

  4. Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

    Directory of Open Access Journals (Sweden)

    Chan Shih-Hung

    2012-07-01

    Full Text Available Abstract Background Insulin receptor substrate (IRS-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3, aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress

  5. Autophagy Alleviates Melamine-Induced Cell Death in PC12 Cells Via Decreasing ROS Level.

    Science.gov (United States)

    Wang, Hui; Gao, Na; Li, Zhigui; Yang, Zhuo; Zhang, Tao

    2016-04-01

    Since melamine was illegally added to raw milk for increasing the apparent protein content, such a scandal has not been quite blown out. Previous studies showed that melamine induced apoptosis and oxidative damage in both in vivo and in vitro experiments. It is well known that autophagy is closely related to oxidative stress. In the present study, we examined whether autophagy played an important role in protecting PC12 cells, which were damaged by melamine. Immunofluorescence assay showed that melamine enhanced the number of punctuate dot, indicating the increase of autophagosomes. Western blot assay presented that melamine significantly elevated the expression level of autophagy markers including LC3-II/LC3-I ratio, beclin-1, and Atg 7. Rapamycin further enhanced the effect, whereas 3-methyadenine (3-MA) inhibited it. MTT assay exhibited that rapamycin significantly enhanced the cell viability (P < 0.01), while 3-MA considerably reduced it in melamine-treated PC12 cells (P < 0.01). Furthermore, flow cytometry assay showed that rapamycin considerably reduced the reactive oxygen species (ROS) level of the cells (P < 0.01), but 3-MA increased the generation of ROS (P < 0.01). Additionally, the superoxide dismutase (SOD) activity was notably increased by rapamycin in melamine-treated PC12 cells (P < 0.01), while the activity of which was prominently decreased by 3-MA (P < 0.01). Malondialdehyde (MDA) assay showed that rapamycin remarkably decreased the MDA level of the cells (P < 0.05), while 3-MA increased it (P < 0.01). Consequently, this study demonstrated that autophagy protected PC12 cells from melamine-induced cell death via inhibiting the excessive generation of ROS. Regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine. PMID:25724280

  6. Deficient autophagy unravels the ROS paradox in chronic granulomatous disease.

    Science.gov (United States)

    van de Veerdonk, Frank L; Dinarello, Charles A

    2014-06-01

    Autophagy defects resulting in inflammation appear to be a key feature in the pathogenesis of Crohn colitis. An inflammatory colitis indistinguishable from Crohn disease is described in patients with chronic granulomatous disease (CGD). Patients with CGD have a mutated NADPH complex and are therefore deficient in reactive oxygen species (ROS) production; however, the underlying mechanism for the inflammatory colitis in CGD remained unknown. In a recent study, our group reported that NADPH-dependent ROS deficiency results in autophagic dysfunction that subsequently contributes to increased IL1B/interleukin 1β production. Mice deficient in the NADPH-complex component NCF4/p40phox, and CGD patients with a defect in NCF4 display minimal recruitment of LC3 to phagosomes in response to internalized bacteria and fungi. Human monocytes from patients with CGD with defective LC3 recruitment show increased IL1B production after LPS stimulation. Blocking IL1 protects NCF4-deficient mice from experimental colitis; importantly, improved clinical outcome in 2 CGD patients with colitis is also observed with IL1 blockade. Moreover, blocking IL1 restores defective autophagy in CGD mice and cells from patients with CGD. Thus, autophagic dysfunction underlies the pathogenesis of granulomatous colitis in CGD, and blocking IL1 can be used to treat CGD colitis. PMID:24879159

  7. Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells.

    Science.gov (United States)

    Martinez, Jennifer; Cunha, Larissa D; Park, Sunmin; Yang, Mao; Lu, Qun; Orchard, Robert; Li, Quan-Zhen; Yan, Mei; Janke, Laura; Guy, Cliff; Linkermann, Andreas; Virgin, Herbert W; Green, Douglas R

    2016-05-01

    Defects in clearance of dying cells have been proposed to underlie the pathogenesis of systemic lupus erythematosus (SLE). Mice lacking molecules associated with dying cell clearance develop SLE-like disease, and phagocytes from patients with SLE often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we and others described a form of noncanonical autophagy known as LC3-associated phagocytosis (LAP), in which phagosomes containing engulfed particles, including dying cells, recruit elements of the autophagy pathway to facilitate maturation of phagosomes and digestion of their contents. Genome-wide association studies have identified polymorphisms in the Atg5 (ref. 8) and possibly Atg7 (ref. 9) genes, involved in both canonical autophagy and LAP, as markers of a predisposition for SLE. Here we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway show increased serum levels of inflammatory cytokines and autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. When dying cells are injected into LAP-deficient mice, they are engulfed but not efficiently degraded and trigger acute elevation of pro-inflammatory cytokines but not anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient, mice accelerated the development of SLE-like disease, including increased serum levels of autoantibodies. By contrast, mice deficient in genes required for canonical autophagy but not LAP do not display defective dying cell clearance, inflammatory cytokine production, or SLE-like disease, and, like wild-type mice, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE. PMID:27096368

  8. Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation

    International Nuclear Information System (INIS)

    Glioblastoma multiforme (GBM) is the most aggressive of the primary brain tumors, with a grim prognosis despite intensive treatment. In the past decades, progress in research has not significantly increased overall survival rate. The in vitro antineoplastic effect and mechanism of action of Casiopeina III-ia (Cas III-ia), a copper compound, on rat malignant glioma C6 cells was investigated. Cas III-ia significantly inhibited cell proliferation, inducing autophagy and apoptosis, which correlated with the formation of autophagic vacuoles, overexpression of LC3, Beclin 1, Atg 7, Bax and Bid proteins. A decrease was detected in the mitochondrial membrane potential and in the activity of caspase 3 and 8, together with the generation of intracellular reactive oxygen species (ROS) and increased activity of c-jun NH2-terminal kinase (JNK). The presence of 3-methyladenine (as selective autophagy inhibitor) increased the antineoplastic effect of Cas III-ia, while Z-VAD-FMK only showed partial protection from the antineoplastic effect induced by Cas III-ia, and ROS antioxidants (N-acetylcysteine) decreased apoptosis, autophagy and JNK activity. Moreover, the JNK –specific inhibitor SP600125 prevented Cas III-ia-induced cell death. Our data suggest that Cas III-ia induces cell death by autophagy and apoptosis, in part due to the activation of ROS –dependent JNK signaling. These findings support further studies of Cas III-ia as candidate for treatment of human malignant glioma

  9. MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch.

    Science.gov (United States)

    Porter, Kristine M; Jeyabalan, Nallathambi; Liton, Paloma B

    2014-06-01

    The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30mmHg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces. PMID:24583119

  10. Degradation of AF1Q by chaperone-mediated autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Huanjie; Cui, Taixing; Li Wang, Xing [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@sdu.edu.cn [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Center for Stem Cell and Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033 (China); Ji, Chunyan, E-mail: jichunyan@sdu.edu.cn [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China)

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  11. Downregulation of B-cell lymphoma/leukemia-2 by overexpressed microRNA 34a enhanced titanium dioxide nanoparticle-induced autophagy in BEAS-2B cells

    Science.gov (United States)

    Bai, Wenlin; Chen, Yujiao; Sun, Pengling; Gao, Ai

    2016-01-01

    Titanium dioxide (TiO2) nanoparticles (TNPs) are manufactured worldwide for a wide range of applications and the toxic effect of TNPs on biological systems is gaining attention. Autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials. MicroRNA 34a (miR34a) acts as a tumor suppressor gene by targeting many oncogenes, but how it affects autophagy induced by TNPs is not completely understood. Here, we observed the activation of TNP-induced autophagy through monodansylcadaverine staining and LC3-I/LC3-II conversion. Meanwhile, the transmission electron microscope ultrastructural analysis showed typical morphological characteristics in autophagy process. We detected the expression of miR34a and B-cell lymphoma/leukemia-2 (Bcl-2). In addition, the underlying mechanism of TNP-induced autophagy was performed using overexpression of miR34a by lentivirus vector transfection. Results showed that TNPs induced autophagy generation evidently. Typical morphological changes in the process of autophagy were observed by the transmission electron microscope ultrastructural analysis and LC3-I/LC3-II conversion increased significantly in TNP-treated cells. Meanwhile, TNPs induced the downregulation of miR34a and increased the expression of Bcl-2. Furthermore, overexpressed miR34a decreased the expression of Bcl-2 both in messenger RNA and protein level, following which the level of autophagy and cell death rate increased after the transfected cells were incubated with TNPs for 24 hours. These findings provide the first evidence that overexpressed miR34a enhanced TNP-induced autophagy and cell death through targeted downregulation of Bcl-2 in BEAS-2B cells.

  12. High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo

    Science.gov (United States)

    Wang, Xiao-Yu; Li, Shuai; Wang, Guang; Ma, Zheng-Lai; Chuai, Manli; Cao, Liu; Yang, Xuesong

    2015-01-01

    High glucose levels induced by maternal diabetes could lead to defects in neural crest development during embryogenesis, but the cellular mechanism is still not understood. In this study, we observed a defect in chick cranial skeleton, especially parietal bone development in the presence of high glucose levels, which is derived from cranial neural crest cells (CNCC). In early chick embryo, we found that inducing high glucose levels could inhibit the development of CNCC, however, cell proliferation was not significantly involved. Nevertheless, apoptotic CNCC increased in the presence of high levels of glucose. In addition, the expression of apoptosis and autophagy relevant genes were elevated by high glucose treatment. Next, the application of beads soaked in either an autophagy stimulator (Tunicamycin) or inhibitor (Hydroxychloroquine) functionally proved that autophagy was involved in regulating the production of CNCC in the presence of high glucose levels. Our observations suggest that the ERK pathway, rather than the mTOR pathway, most likely participates in mediating the autophagy induced by high glucose. Taken together, our observations indicated that exposure to high levels of glucose could inhibit the survival of CNCC by affecting cell apoptosis, which might result from the dysregulation of the autophagic process. PMID:26671447

  13. Dihydromyricetin improves skeletal muscle insulin resistance by inducing autophagy via the AMPK signaling pathway.

    Science.gov (United States)

    Shi, Linying; Zhang, Ting; Liang, Xinyu; Hu, Qin; Huang, Juan; Zhou, Yong; Chen, Mingliang; Zhang, Qianyong; Zhu, Jundong; Mi, Mantian

    2015-07-01

    Skeletal muscle insulin resistance (SMIR) plays an important role in the pathogenesis of type 2 diabetes. Dihydromyricetin (DHM), a natural flavonoid, exerts various bioactivities including anti-oxidative and hepatoprotective effects. Herein, we intended to determine the effect of DHM on SMIR and the underlying mechanisms. We found that DHM increased the expression of phosphorylated insulin receptor substrate-1, phosphorylated Akt and glucose uptake capacity in palmitate-treated L6 myotubes under insulin-stimulated conditions. The expression of light chain 3, Beclin 1, autophagy-related gene 5 (Atg5), the degradation of sequestosome 1 and the formation of autophagosomes were also upregulated by DHM. Suppression of autophagy by 3-methyladenine and bafilomycin A1 or Atg5 and Beclin1 siRNA abolished the favorable effects of DHM on SMIR. Furthermore, DHM increased the levels of phosphorylated AMP-activated protein kinase (AMPK) and Ulk1, and decreased phosphorylated mTOR levels. AMPK inhibitor compound C (CC) and AMPK siRNA abrogated DHM-induced autophagy, subsequently suppressed DHM-induced SMIR improvement. Additionally, DHM inhibited the activity of F1F0-ATPase thereby activating AMPK. Finally, the results of in vivo study conducted in high fat diet-fed rats were consistent with the findings of in vitro study. In conclusion, DHM improved SMIR by inducing autophagy via the activation of AMPK signaling pathway. PMID:25797177

  14. Autophagy and cellular senescence mediated by Sox2 suppress malignancy of cancer cells.

    Directory of Open Access Journals (Sweden)

    Yong-Yeon Cho

    Full Text Available Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the "stemness" of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16(INK4a, p21 and phosphorylated p53 (Ser15. Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

  15. 自噬过程的晚期阶段%The Late Stages of Autophagy

    Institute of Scientific and Technical Information of China (English)

    杜万清; 俞立

    2011-01-01

    自噬是高度保守的细胞内降解途径.在此过程中,部分细胞质和细胞器被双层膜的囊泡包裹形成自噬体,随后与溶酶体融合并降解被吞噬的物质.降解产物被释放到细胞质中重新用于必需的物质和能量合成.本文主要关注自噬的晚期阶段,即从自噬体合成结束到溶酶体再生过程.通过对这一过程相关基因及蛋白产物的研究,初步揭示了此过程的分子机制.%Autophagy is highly conserved degradation process in which portions of cytosol and organelles are sequestered into a double-membrane vesicle, an autophagosome, and delivered into a degradative organelle, the vacuole/lysosome, for breakdown and eventual recycling of the resulting macromolecules. In this review, we discussed the late stages of autophagy, such as fusion of autophagosome with lysosome/vacuole, degradation of autophagosome and reformation of lysosome from hybrids. Identification of many autophagy-related genes reveals the machinery of the late stages of autophagy. Here, we summarize current information about the molecular mechanism of this process initially.

  16. Human stefin B role in cell's response to misfolded proteins and autophagy.

    Directory of Open Access Journals (Sweden)

    Mira Polajnar

    Full Text Available Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

  17. Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy

    Science.gov (United States)

    Pawar, Kamlesh; Hanisch, Carlos; Palma Vera, Sergio Eliseo; Einspanier, Ralf; Sharbati, Soroush

    2016-01-01

    Small non-coding RNA play a major part in host response to bacterial agents. However, the role of long non-coding RNA (lncRNA) in this context remains unknown. LncRNA regulate gene expression by acting e.g. as transcriptional coactivators, RNA decoys or microRNA sponges. They control development, differentiation and cellular processes such as autophagy in disease conditions. Here, we provide an insight into the role of lncRNA in mycobacterial infections. Human macrophages were infected with Mycobacterium bovis BCG and lncRNA expression was studied early post infection. For this purpose, lncRNA with known immune related functions were preselected and a lncRNA specific RT-qPCR protocol was established. In addition to expression-based prediction of lncRNA function, we assessed strategies for thorough normalisation of lncRNA. Arrayed quantification showed infection-dependent repression of several lncRNA including MEG3. Pathway analysis linked MEG3 to mTOR and PI3K-AKT signalling pointing to regulation of autophagy. Accordingly, IFN-γ induced autophagy in infected macrophages resulted in sustained MEG3 down regulation and lack of IFN-γ allowed for counter regulation of MEG3 by viable M. bovis BCG. Knockdown of MEG3 in macrophages resulted in induction of autophagy and enhanced eradication of intracellular M. bovis BCG. PMID:26757825

  18. Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/reperfusion induced apoptosis and autophagy in the rat

    Directory of Open Access Journals (Sweden)

    Chien Chiang-Ting

    2009-02-01

    Full Text Available Abstract Prolonged ischemia amplified iscehemia/reperfusion (IR induced renal apoptosis and autophagy. We hypothesize that ischemic conditioning (IC by a briefly intermittent reperfusion during a prolonged ischemic phase may ameliorate IR induced renal dysfunction. We evaluated the antioxidant/oxidant mechanism, autophagy and apoptosis in the uninephrectomized Wistar rats subjected to sham control, 4 stages of 15-min IC (I15 × 4, 2 stages of 30-min IC (I30 × 2, and total 60-min ischema (I60 in the kidney followed by 4 or 24 hours of reperfusion. By use of ATP assay, monitoring O2-. amounts, autophagy and apoptosis analysis of rat kidneys, I60 followed by 4 hours of reperfusion decreased renal ATP and enhanced reactive oxygen species (ROS level and proapoptotic and autophagic mechanisms, including enhanced Bax/Bcl-2 ratio, cytochrome C release, active caspase 3, poly-(ADP-ribose-polymerase (PARP degradation fragments, microtubule-associated protein light chain 3 (LC3 and Beclin-1 expression and subsequently tubular apoptosis and autophagy associated with elevated blood urea nitrogen and creatinine level. I30 × 2, not I15 × 4 decreased ROS production and cytochrome C release, increased Manganese superoxide dismutase (MnSOD, Copper-Zn superoxide dismutase (CuZnSOD and catalase expression and provided a more efficient protection than I60 against IR induced tubular apoptosis and autophagy and blood urea nitrogen and creatinine level. We conclude that 60-min renal ischemia enhanced renal tubular oxidative stress, proapoptosis and autophagy in the rat kidneys. Two stages of 30-min ischemia with 3-min reperfusion significantly preserved renal ATP content, increased antioxidant defense mechanisms and decreased ischemia/reperfusion enhanced renal tubular oxidative stress, cytosolic cytochrome C release, proapoptosis and autophagy in rat kidneys.

  19. Exercise induces autophagy in peripheral tissues and in the brain

    OpenAIRE

    He, Congcong; Sumpter, Jr., Rhea; Levine, Beth

    2012-01-01

    We recently identified physical exercise as a newly defined inducer of autophagy in vivo. Exercise induced autophagy in multiple organs involved in metabolic regulation, such as muscle, liver, pancreas and adipose tissue. To study the physiological role of exercise-induced autophagy, we generated mice with a knock-in nonphosphorylatable mutation in BCL2 (Thr69Ala, Ser70Ala and Ser84Ala) (BCL2 AAA) that are defective in exercise- and starvation-induced autophagy but not in basal autophagy. We ...

  20. 他克莫司通过上调自噬作用保护2型糖尿病大鼠足细胞%Tacrolimus protects podocytes by up-regulating autophagy in type 2 diabetic model rats

    Institute of Scientific and Technical Information of China (English)

    王彤; 马瑞霞; 武国华; 孙益婷

    2016-01-01

    Objective To assess the effects of tacrolimus (FKS06) on podocyte in type 2 diabetic model rats and to explore the potential mechanism.Methods The model rats were fed with high fat and high sugar food and combining with a low-dose of streptozotocin (STZ).They were then randomly divided into a diabetic mellitus group (DM group) and a FK506 group.A normal control group (NC group) was also set.The rats in FK506 group were given with 0.5 mg· kg-1· d-1 FK506 for 8 weeks.The biochemical parameters were measured.The changes of renal pathology and ultrastructure of podocyte were observed by the light and electron microscopy.The expression of nephrin and LC3-Ⅱ was determined by immunohistochemistry and Western blotting.Results (1) Compared with those in NC group,KW/BW,systolic blood pressure (SBP),fasting blood glucose (FBG),triglyceride (TG),total cholesterol (TC),urinary albumin excretion rate (UAE) and creatinine clearance rate (Ccr) in DM group were significantly increased (all P < 0.05).And the KW/BW,UAE and Ccr were decreased in FK506 group compared to those in DM group (all P < 0.05),while other parameters had no significant difference (all P > 0.05).(2) Compared with those in NC group,the glomerular volume,mesangial cell proliferation and accumulation of mesangial matrix were increased,and the foot process became disorder and fusion in DM group,while these changes were significantly reduced in FK506 group.(3) Compared with that in NC group,the expression of nephrin and LC3-Ⅱ was decreased in DM group (all P < 0.05),and both of parameters were higher in FK506 group than those in DM group (all P < 0.05).Conclusion FK506 may enhance podocyte autophagy in type 2 diabetic model rats and attenuate podocyte injury.%目的 探讨他克莫司(FK506)对2型糖尿病(T2DM)大鼠足细胞的保护作用及其可能机制.方法 高糖高脂喂养联合小剂量链佐星(STZ)构建T2DM大鼠模型,模型大鼠随机分为糖尿病组(DM组)和FK506

  1. Autophagy in the light of sphingolipid metabolism

    DEFF Research Database (Denmark)

    Harvald, Eva Bang; Olsen, Anne Sofie Braun; Færgeman, Nils J.

    2015-01-01

    Maintenance of cellular homeostasis requires tight and coordinated control of numerous metabolic pathways, which are governed by interconnected networks of signaling pathways and energy-sensing regulators. Autophagy, a lysosomal degradation pathway by which the cell self-digests its own components......, has over the past decade been recognized as an essential part of metabolism. Autophagy not only rids the cell of excessive or damaged organelles, misfolded proteins, and invading microorganisms, it also provides nutrients to maintain crucial cellular functions. Besides serving as essential structural...... moieties of biomembranes, lipids including sphingolipids are increasingly being recognized as central regulators of a number of important cellular processes, including autophagy. In the present review we describe how sphingolipids, with special emphasis on ceramides and sphingosine-1-phosphate, can act as...

  2. Lipopolysaccharide induction of autophagy is associated with enhanced bactericidal activity in Dictyostelium discoideum

    OpenAIRE

    Pflaum, Katherine; Gerdes, Kimberly; Yovo, Kossi; Callahan, Jennifer; Snyder, Michelle L.D.

    2012-01-01

    Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum ce...

  3. What to Eat: Evidence for Selective Autophagy in Plants

    Institute of Scientific and Technical Information of China (English)

    Brice E.Floyd; Stephanie C.Morriss; Gustavo C.Maclntosh; Diane C.Bassham

    2012-01-01

    Autophagy is a macromolecular degradation pathway by which cells recycle their contents as a developmental process,house-keeping mechanism,and response to environmental stress.In plants,autophagy involves the sequestration of cargo to be degraded,transport to the cell vacuole in a double-membrane bound autophagosome,and subsequent degradation by lytic enzymes.Autophagy has generally been considered to be a non-selective mechanism of degradation.However,studies in yeast and animals have found numerous examples of selective autophagy,with cargo including proteins,protein aggregates,and organelles.Recent work has also provided evidence for several types of selective autophagy in plants.The degradation of protein aggregates was the first selective autophagy described in plants,and,more recently,a hybrid protein of the mammalian selective autophagy adaptors p62 and NBR1,which interacts with the autophagy machinery and may function in autophagy of protein aggregates,was described in plants.Other intracellular components have been suggested to be selectively targeted by autophagy in plants,but the current evidence is limited.Here,we discuss recent findings regarding the selective targeting of cell components by autophagy in plants.

  4. Avian influenza A virus H5N1 causes autophagy-mediated cell death through suppression of mTOR signaling

    Institute of Scientific and Technical Information of China (English)

    Jianhui Ma; Qian Sun; Ruifang Mi; Hongbing Zhang

    2011-01-01

    Of the few avian influenza viruses that have crossed the species barrier to infect humans,the highly pathogenic influenza A (H5N1) strain has claimed the lives of more than half of the infected patients.With largely unknown mechanism of lung injury by H5N1 infection,acute respiratory distress syndrome (ARDS) is the major cause of death among the victims.Here we present the fact that H5N1 caused autophagic cell death through suppression of mTOR signaling.Inhibition of autophagy,either by depletion of autophagy gene Beclinl or by autophagy inhibitor 3-methyladenine (3-MA),significantly reduced H5N1 mediated cell death.We suggest that autophagic cell death may contribute to the development of ARDS in H5N1 influenza patients and inhibition of autophagy could therefore become a novel strategy for the treatment of H5N1 infection.

  5. Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in C. elegans

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Kruse, Rikke; Harvald, Eva Bang; Olsen, Anne Sofie Braun; Gallego, Sandra Fernandez; Hannibal-Bach, Hans Kristian; Ejsing, Christer S.; Færgeman, Nils J

    2013-01-01

    Ceramide and its metabolites constitute a diverse group of lipids, which play important roles as structural entities of biological membranes as well as regulators of cellular growth, differentiation, and development. The C. elegans genome comprises three ceramide synthase genes; hyl-1, hyl-2, and...... lagr-1. HYL-1 function is required for synthesis of ceramides and sphingolipids containing very long acyl-chains (≥C24), while HYL-2 is required for synthesis of ceramides and sphingolipids containing shorter acyl-chains (≤C22). Here we show that functional loss of HYL-2 decreases lifespan, while loss...... of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also...

  6. Plac8 Links Oncogenic Mutations to Regulation of Autophagy and Is Critical to Pancreatic Cancer Progression

    Directory of Open Access Journals (Sweden)

    Conan Kinsey

    2014-05-01

    Full Text Available Mutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

  7. Induction of autophagy improves embryo viability in cloned mouse embryos

    Science.gov (United States)

    Shen, XingHui; Zhang, Na; Wang, ZhenDong; Bai, GuangYu; Zheng, Zhong; Gu, YanLi; Wu, YanShuang; Liu, Hui; Zhou, DongJie; Lei, Lei

    2015-01-01

    Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT. PMID:26643778

  8. Urocortin抑制心肌缺血再灌注诱导的自噬%Urocortin inhibits myocardium ischemia/reperfusion-induced autophagy

    Institute of Scientific and Technical Information of China (English)

    张冠鑫; 韩林; 郭志福; 王崇; 任安经; 钟铿; 李鑫; 袁扬; 徐志云

    2013-01-01

    目的 研究uroeortin (UCN)对缺血再灌注诱导的心肌细胞自噬的影响,探讨UCN的心肌保护机制.方法 构建大鼠在体心脏缺血再灌注损伤和离体新生大鼠心肌细胞的缺氧复氧模型,进行缺血/缺氧1h再灌注/复氧2h的损伤,在缺血/缺氧前1h给予UCN预处理;在再灌注/复氧2h后观察UCN对缺血再灌注/缺氧复氧诱导的心肌损伤、细胞自噬和自噬相关基因表达的影响.结果 UCN预处理可以显著降低缺血再灌注损伤导致的心肌损害,使梗死面积降低,血清肌酸激酶(CK)、乳酸脱氢酶(LDH)降低;增加缺氧复氧离体心肌细胞活力、减少培养上清的LDH水平.与上述心肌保护作用相伴随的是UCN预处理还能抑制缺氧复氧导致的心肌细胞自噬,使LC3BⅡ/LC3BI的比值显著降低,并且抑制自噬相关基因Beclin1、Bni p3的mRNA表达.结论 UCN可以抑制缺血再灌注诱导的心肌细胞自噬,可能在抗缺血再灌注损伤中起重要作用.%Objective To study the regulatory effect of urocortin (UCN) on ischemia/reperfusion (I/R)-induced myocardial autophagy,so as to explore the myocardial protection mechanism of UCN.Methods Cardiac I/R model was established with rats and hypoxia/reoxygenation(H/R) model was also established with neonatal rat cardiomyocytes.The injury was created by ischemic/hypoxia for 1 h plus reperfusion/reoxygenation for 2 h,and UCN pretreatment was given 1 h before ischemia/hypoxia.The I/R or H/R-induced myocardial injury,myocardial autophagy and autophagy-related gene expression were observed 2 h after reperfusion/reoxygenation.Results UCN pretreatment greatly reduced I/R-induced myocardial damage by decreasing the infarct size,serum creatine kinase (CK) and lactate dehydrogenase (LDH) concentration,increasing the vitality of H/R cardiomyocytes in vitro,and reducing LDH level in the culture supernatant.Moreover,UCN pretreatment also inhibited H/R-induced myocardial autophagy by reducing the ratio

  9. Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1.

    Science.gov (United States)

    Ge, Rui; Liu, Lin; Dai, Wei; Zhang, Weigang; Yang, Yuqi; Wang, Huina; Shi, Qiong; Guo, Sen; Yi, Xiuli; Wang, Gang; Gao, Tianwen; Luan, Qi; Li, Chunying

    2016-06-01

    Xeroderma pigmentosum group A (XPA), a key protein in the nucleotide excision repair pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance of melanoma to platinum-based drugs like cisplatin is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) activation and that the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. Finally, we proved that the inhibition of PARP1 and the autophagy process made resistant melanoma cells more susceptible to cisplatin treatment. Our study shows that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin and that the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy. PMID:26880244

  10. Autophagy Has a Beneficial Role in Relieving Cigarette Smoke-Induced Apoptotic Death in Human Gingival Fibroblasts

    Science.gov (United States)

    Kim, Moon-Soo; Yun, Jeong-Won; Park, Jin-Ho; Park, Bong-Wook; Kang, Young-Hoon; Hah, Young-Sool; Hwang, Sun-Chul; Woo, Dong Kyun; Byun, June-Ho

    2016-01-01

    The deleterious role of cigarette smoke has long been documented in various human diseases including periodontal complications. In this report, we examined this adverse effect of cigarette smoke on human gingival fibroblasts (HGFs) which are critical not only in maintaining gingival tissue architecture but also in mediating immune responses. As well documented in other cell types, we also observed that cigarette smoke promoted cellular reactive oxygen species in HGFs. And we found that this cigarette smoke-induced oxidative stress reduced HGF viability through inducing apoptosis. Our results indicated that an increased Bax/Bcl-xL ratio and resulting caspase activation underlie the apoptotic death in HGFs exposed to cigarette smoke. Furthermore, we detected that cigarette smoke also triggered autophagy, an integrated cellular stress response. Interesting, a pharmacological suppression of the cigarette smoke-induced autophagy led to a further reduction in HGF viability while a pharmacological promotion of autophagy increased the viability of HGFs with cigarette smoke exposures. These findings suggest a protective role for autophagy in HGFs stressed with cigarette smoke, highlighting that modulation of autophagy can be a novel therapeutic target in periodontal complications with cigarette smoke.

  11. DT-13, a saponin monomer of dwarf lilyturf tuber, induces autophagy and potentiates anti-cancer effect of nutrient deprivation.

    Science.gov (United States)

    Li, Hongyang; Sun, Li; de Carvalho, Evandro Lopes; Li, Xinxin; Lv, Xiaodan; Khan, Ghulam Jilany; Semukunzi, Herve; Yuan, Shengtao; Lin, Sensen

    2016-06-15

    Metabolic stress induces autophagy as a protective mechanism in tumorigenesis and development. Conversely, excessive autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy. DT-13, the saponin monomer 13 of the Dwarf lilyturf tuber, inhibited tumor metastasis and angiogenesis in previous studies. However, there is scarcity of data regarding the effect of DT-13 on autophagy process. Here, we demonstrated that DT-13 induced autophagy in human cancer cell lines and caused significant cell apoptosis under nutrient starvation. We firstly showed that DT-13 increased the accumulation of GFP-LC3 puncta and induced the expression of LC3-II in a dose- and time-dependent manner. DT-13 also upregulated the expression of Beclin-1, Atg-3 and Atg-7, and induced autophagic flux in human gastric cancer BGC-823 cells. We next found that low-toxic concentrations of DT-13 significantly induced apoptosis under nutrient deprivation. We finally demonstrated that the PI3K/Akt/mTOR signal pathway was involved in the cytotoxic effect of DT-13. Our data indicated that DT-13 was a novel autophagy inducer and might be considered in future treatment of cancer. PMID:27079642

  12. Adenovirus-mediated wild-type PTEN promoting glioma stem/progenitor cells autophagy activity

    OpenAIRE

    ZHAO Yao-dong; Zi-long WEI; Zhang, Quan-Bin; LOU Mei-qing; HUANG, QIANG

    2013-01-01

    Background PTEN is an anti-oncogene frequently inactivating in glioma. The previous study found that PTEN was closely related to cellular autophagy activity. The purpose of this paper is to study whether the inactivation of PTEN in glioma stem/progenitor cells (GSPCs) is correlative with the low autophagic activity in GSPCs. Methods Wild-type PTEN genes were transferred into GSPCs mediated by adenovirus. The autophagic activity in GSPCs before or after the introduction of wild-type PTEN was...

  13. Establishing a novel C. elegans model to investigate the role of autophagy in amyotrophic lateral sclerosis

    OpenAIRE

    Li, Jia; Huang, Kai-xing; Le, Wei-Dong

    2013-01-01

    Aim: To develop a C. elegans model of amyotrophic lateral sclerosis (ALS) and to evaluate the role of autophagy in the disease. Methods: Stable transgenic worms expressing the G93A mutant form of Cu,Zn-superoxide dismutase (SOD1) in GABAergic motor neurons were generated. Axon guidance and protein aggregation in the motor neurons were observed with fluorescence microscopy. A paralysis assay was performed to evaluate the motor function of the transgenic worms. The expression of autophagic gene...

  14. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of); Kim, Young Woo; Cho, Il Je; Kim, Sang Chan [Medical Research Center for Globalization of Herbal Formulation, College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715 (Korea, Republic of); Shin, Sang Mi [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of); Ki, Sung Hwan, E-mail: shki@chosun.ac.kr [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of)

    2014-01-15

    Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

  15. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    International Nuclear Information System (INIS)

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs

  16. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yoko [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Sou, Yu-Shin; Kageyama, Shun [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Takahashi, Takao [Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Ueno, Takashi [Division of Proteomics and Biomolecular Science, Center for Biomedical Research Resources, Juntendo University Graduate School of Medicine, Tokyo 113-8421 (Japan); Tanaka, Keiji [Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Komatsu, Masaaki, E-mail: komatsu-ms@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Biochemistry, School of Medicine, Niigata University, Niigata 951-8510 (Japan); Ichimura, Yoshinobu, E-mail: ichimura-ys@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan)

    2014-03-28

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.

  17. Fibrates inhibit the apoptosis of Batten disease lymphoblast cells via autophagy recovery and regulation of mitochondrial membrane potential.

    Science.gov (United States)

    Hong, Minho; Song, Ki Duk; Lee, Hak-Kyo; Yi, SunShin; Lee, Yong Seok; Heo, Tae-Hwe; Jun, Hyun Sik; Kim, Sung-Jo

    2016-03-01

    Batten disease (BD; also known as juvenile neuronal ceroid lipofuscinosis) is a genetic disorder inherited as an autosomal recessive trait and is characterized by blindness, seizures, cognitive decline, and early death resulting from the inherited mutation of the CLN3 gene. Mitochondrial oxidative stress, endoplasmic reticulum (ER) stress, disrupted autophagy, and enhanced apoptosis have been suggested to play a role in BD pathogenesis. Fibrates, a class of lipid-lowering drugs that induce peroxisome proliferator-activated receptor-α (PPAR-α) activation, are the most commonly used PPAR agonists. Assuming that fibrates have a neuroprotective effect, we studied the effects of fibrates, fenofibrate, bezafibrate, and gemfibrozil on apoptosis, depolarization of mitochondrial membrane, and defective autophagy in BD lymphoblast cells. The viability of fibrate-treated BD lymphoblast cells increased to levels of normal lymphoblast cells. In addition, treatment with fibrates inhibited depolarization of mitochondrial membrane potential in BD lymphoblast cells. Defective autophagy in BD lymphoblast cells was normalized when treated with fibrates as indicated by increased acridine orange staining. The recovery of autophagy in BD lymphoblast cells is most likely attributed to the upregulation of autophagy proteins, lysosomal-associated membrane protein 1 (LAMP1), and LC3 I/II, after treatment with fibrates. This study therefore suggests that fibrates may have a therapeutic potential against BD. PMID:26659390

  18. Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

    International Nuclear Information System (INIS)

    Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H2O2 production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling

  19. Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling, E-mail: shanglingwang@126.com

    2015-07-01

    Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H{sub 2}O{sub 2} production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling.

  20. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

    Directory of Open Access Journals (Sweden)

    D.Y. Xia

    2013-08-01

    Full Text Available Sublethal ischemic preconditioning (IPC is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration, 5×5 min (5 min duration, 2 episodes, 15-min interval, 5×5×5 min (5 min duration, 3 episodes, 15-min intervals, and 15 min (15 min duration, and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

  1. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Xia, D.Y. [Department of Neurology, Navy General Hospital of PLA, Beijing (China); Li, W. [General Hospital of Shenyang Military Command, Department of Neurology, Shenyang, China, Department of Neurology, General Hospital of Shenyang Military Command, Shenyang (China); Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K. [Department of Neurology, Navy General Hospital of PLA, Beijing (China)

    2013-08-10

    Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

  2. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

    International Nuclear Information System (INIS)

    Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis

  3. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

    International Nuclear Information System (INIS)

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  4. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  5. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Ping-Ge [Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Zhi-Xin [Centre Laboratory, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China); Li, Jian-Hua [Department of Geriatric Cardiology, Chinese PLA General Hosptial, Beijing 100853 (China); Zhou, Zhe, E-mail: zhouzhe76@126.com [Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Zhang, Qing-Hua, E-mail: 1056055170@qq.com [Department of Cardiology, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China)

    2015-08-07

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

  6. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

    International Nuclear Information System (INIS)

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival

  7. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Bolt, Alicia M.; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T., E-mail: klimecki@pharmacy.arizona.edu

    2012-10-15

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  8. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    International Nuclear Information System (INIS)

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility

  9. Intrastriatal GDNF gene transfer by inducible lentivirus vectors protects dopaminergic neurons in a rat model of parkinsonism.

    Science.gov (United States)

    Chen, Sha-Sha; Yang, Chun; Hao, Fei; Li, Chen; Lu, Tao; Zhao, Li-Ru; Duan, Wei-Ming

    2014-11-01

    Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects on dopaminergic (DA) neurons both in vivo and in vitro. However, substantial evidence has shown that a long-term overexpression of GDNF gene is often associated with side effects. We previously improved tetracycline (Tet)-On lentivirus system carrying human GDNF (hGDNF) gene, and demonstrated that hGDNF gene expression was tightly regulated and functional in vitro. Here we further examined the efficiency and neuroprotection of Tet-On lentivirus-mediated hGDNF gene regulation in neural progenitor cells (NPCs) and a rat model of parkinsonism. The results showed that hGDNF gene expression was tightly regulated in transduced NPCs. Doxycycline (Dox)-induced hGDNF protected DA neurons from 6-hydroxydopamine (6-OHDA)-induced toxicity in vitro. Intrastriatal injections of Tet-On lentivirus vectors resulted in dramatically increased levels of hGDNF protein in the striatum of rats with Dox-drinking water, when compared to lentivirus-injected and saline-injected rats with normal drinking water, respectively. In addition, hGDNF protected nigral DA neurons and striatal DA fibers, and attenuated d-amphetamine-induced rotational asymmetry in the 6-OHDA lesioned rats. To the best of our knowledge, this is the first report that hGDNF gene transfer by Tet-On lentivirus vectors is tightly regulated in rat brain, and Dox-induced hGDNF is functional in neuroprotection of nigral DA neurons in a rat model of parkinsonism. PMID:24997241

  10. Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy.

    Science.gov (United States)

    Moore, Claire E J; Wang, Xuemin; Xie, Jianling; Pickford, Jo; Barron, John; Regufe da Mota, Sergio; Versele, Matthias; Proud, Christopher G

    2016-04-01

    Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy. PMID:26795954

  11. Autophagy as a Potential Target for Sarcopenia.

    Science.gov (United States)

    Fan, Jingjing; Kou, Xianjuan; Jia, Shaohui; Yang, Xiaoqi; Yang, Yi; Chen, Ning

    2016-07-01

    Sarcopenia is an aging-related disease with a significant reduction in mass and strength of skeletal muscle due to the imbalance between protein synthesis and protein degradation. The loss of skeletal muscle is an inevitable event during aging process, which can result in the significant impact on the quality of life, and also can increase the risk for other aging-associated diseases in the elderly. However, the underlying molecular mechanism of aging-related skeletal muscle loss is still poorly understood. Autophagy is a degradation pathway for the clearance of dysfunctional organelles and damaged macromolecules during aging process. Appropriate induction or accurate regulation of autophagic process and improved quality control of mitochondria through autophagy or other strategies are required for the maintenance of skeletal muscle mass. In this article, we have summarized the current understanding of autophagic pathways in sarcopenia, and discussed the functional status of autophagy and autophagy-associated quality control of mitochondria in the pathogenesis of sarcopenia. Moreover, this article will provide some theoretical references for the exploration of scientific and optimal intervention strategies such as exercise and caloric restriction for the prevention and treatment of sarcopenia through the regulation of autophagic pathways. PMID:26580995

  12. The thiazole derivative CPTH6 impairs autophagy.

    Science.gov (United States)

    Ragazzoni, Y; Desideri, M; Gabellini, C; De Luca, T; Carradori, S; Secci, D; Nescatelli, R; Candiloro, A; Condello, M; Meschini, S; Del Bufalo, D; Trisciuoglio, D

    2013-01-01

    We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in

  13. Impairment of autophagy: From hereditary disorder to drug intoxication

    International Nuclear Information System (INIS)

    At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinson's, Alzheimer's, and Huntington's diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication

  14. Fine pathogen discrimination within the APL1 gene family protects Anopheles gambiae against human and rodent malaria species.

    Directory of Open Access Journals (Sweden)

    Christian Mitri

    2009-09-01

    Full Text Available Genetically controlled resistance of Anopheles gambiae mosquitoes to Plasmodium falciparum is a common trait in the natural population, and a cluster of natural resistance loci were mapped to the Plasmodium-Resistance Island (PRI of the A. gambiae genome. The APL1 family of leucine-rich repeat (LRR proteins was highlighted by candidate gene studies in the PRI, and is comprised of paralogs APL1A, APL1B and APL1C that share > or =50% amino acid identity. Here, we present a functional analysis of the joint response of APL1 family members during mosquito infection with human and rodent Plasmodium species. Only paralog APL1A protected A. gambiae against infection with the human malaria parasite P. falciparum from both the field population and in vitro culture. In contrast, only paralog APL1C protected against the rodent malaria parasites P. berghei and P. yoelii. We show that anti-P. falciparum protection is mediated by the Imd/Rel2 pathway, while protection against P. berghei infection was shown to require Toll/Rel1 signaling. Further, only the short Rel2-S isoform and not the long Rel2-F isoform of Rel2 confers protection against P. falciparum. Protection correlates with the transcriptional regulation of APL1A by Rel2-S but not Rel2-F, suggesting that the Rel2-S anti-parasite phenotype results at least in part from its transcriptional control over APL1A. These results indicate that distinct members of the APL1 gene family display a mutually exclusive protective effect against different classes of Plasmodium parasites. It appears that a gene-for-pathogen-class system orients the appropriate host defenses against distinct categories of similar pathogens. It is known that insect innate immune pathways can distinguish between grossly different microbes such as Gram-positive bacteria, Gram-negative bacteria, or fungi, but the function of the APL1 paralogs reveals that mosquito innate immunity possesses a more fine-grained capacity to distinguish between

  15. Autophagy counteracts apoptosis in human multiple myeloma cells exposed to oridonin in vitro via regulating intracellular ROS and SIRT1

    Institute of Scientific and Technical Information of China (English)

    Rong ZENG; Yan CHEN; Shuai ZHAO; Guo-hui CUI

    2012-01-01

    To explore the mechanisms underlying the oridonin-induced apoptosis and autophagy in human multiple myeloma cells in vitro.Methods:Human multiple myeloma RPMI8266 cells were used.The cell viability was assessed using MTT assay.Morphological changes of apoptosis and autophagy were observed under transmission electron microscope.TUNEL and annexin V-FITC/PI dual staining assays were used to measure apoptosis.Autophagy was analyzed using Western blot analysis and immunofluorescence staining with a QDs605 nm-Anti-LC3 fluorescent probe.Intracellular ROS was estimated with flow cytometry using DCFH-DA fluorescent probe.Protein levels of active caspase 3,Beclin 1 and SIRT1 were determined with Western blot analysis.Results:Exposure to oridonin (1-64 μmol/L) inhibited the proliferation of RPMI8266 cells in a concentration-dependent manner with an IC50 value of 6.74 μmol/L.Exposure to oridonin (7 μmol/L) simultaneously induced caspase 3-mediated apoptosis and Beclin 1-dependent autophagy of RPMI8266 cells.Both the apoptosis and autophagy were time-dependent,and apoptosis was the main effector pathway of cell death.Exposure to oridonin (7 μmol/L) increased intracellular ROS and reduced SIRT1 nuclear protein in a time-dependent manner.The blockade of intracellular generation of ROS by NAC (5 mmol/L) abrogated apoptosis,autophagy and the decrease of SIRT1 in the cells exposed to oridonin (7 μmol/L).The inhibition of autophagy by 3-MA (5 mmol/L) sensitized the cells to oridonin-induced apoptosis,which was accompanied by increased intracellular ROS and decreased SlRT1.Conclusion:Oridonin simultaneously induces apoptosis and autophagy of human multiple myeloma RPMI8266 cells via regulation of intracellular ROS generation and SIRT1 nuclear protein.The cytotoxicity of oridonin is mainly mediated through the apoptotic pathway,whereas the autophagy protects the cells from apoptosis.

  16. Induction of cytoprotective autophagy in PC-12 cells by cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Qiwen [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China); Bijie Pilot Area Research Institute of Bijie University, Bijie 551700 (China); Zhu, Jiaqiao; Zhang, Kangbao; Jiang, Chenyang; Wang, Yi; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China)

    2013-08-16

    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.

  17. Induction of cytoprotective autophagy in PC-12 cells by cadmium

    International Nuclear Information System (INIS)

    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity

  18. Melatonin antagonizes cadmium-induced neurotoxicity by activating the transcription factor EB-dependent autophagy-lysosome machinery in mouse neuroblastoma cells.

    Science.gov (United States)

    Li, Min; Pi, Huifeng; Yang, Zhiqi; Reiter, Russel J; Xu, Shangcheng; Chen, Xiaowei; Chen, Chunhai; Zhang, Lei; Yang, Min; Li, Yuming; Guo, Pan; Li, Gaoming; Tu, Manyu; Tian, Li; Xie, Jia; He, Mindi; Lu, Yonghui; Zhong, Min; Zhang, Yanwen; Yu, Zhengping; Zhou, Zhou

    2016-10-01

    Cadmium (Cd), a highly ubiquitous heavy metal, induces neurotoxicity. Melatonin, a major secretory product of the pineal gland, protects against Cd-induced neurotoxicity. However, the mechanism that accounts for this protection remains to be elucidated. Herein, we exposed mouse neuroblastoma cells (Neuro-2a cells) to different concentrations of cadmium chloride (CdCl2 ) (12.5, 25, and 50 μ mol L(-1) ) for 24 hours. We showed that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function, subsequently leading to nerve cell death. In addition, Cd decreases the level of transcription factor EB (TFEB) but induces the nuclear translocation of TFEB, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Moreover, compared to the 50-μ mol L(-1) Cd group, administration of 1 μ mol L(-1) melatonin increased "TFEB-responsive genes" (Pfusion (0.05±0.00 vs 0.21±0.01, Pnuclear translocation (2.81±0.08 vs 3.82±0.05, P<.05). Tfeb siRNA blocked the melatonin-mediated elevation in autophagy-lysosome machinery in Cd-induced neurotoxicity (P<.01). Taken together, these results uncover a potent role for TFEB-mediated autophagy in the pathogenesis of Cd-induced neurotoxicity, suggesting that control of the autophagic pathway by melatonin might provide an important clue for exploring potential targets for novel therapeutics of Cd-induced neurotoxicity. PMID:27396692

  19. 'Cancer genes and their expression; blueprint to future improvements in radiation protection?'

    International Nuclear Information System (INIS)

    The field of molecular oncology is reviewed, with some reference to the effects of ionizing radiation. There are three classes of cancer genes: oncogenes, tumor suppressor genes, and modulator genes. Proto-oncogenes control the synthesis of proteins which regulate cell division; these proto-oncogenes, of which about 100 have been identified, become oncogenes when damaged. Oncogenes are dominant. Only about a dozen tumor-suppressor genes have been identified; they are recessive. One example of a tumor suppressor gene is that producing p53 protein, which shuts down DNA replication temporarily to allow time for enzymatic repair of damage resulting from radiation or other damaging agents. Modulator genes are a heterogeneous class, including those genes that facilitate the destruction of pre-malignant cells by immune surveillance. Modulator genes also include those responsible for DNA repair. The study of DNA repair and its defects has made recent strides, and 12 participating enzymes have now been identified. It may soon be possible to monitor low-level radiation effects in individuals at the molecular level. Perhaps individuals will be monitored for their 'carcinogen exposure response' index, giving them the opportunity to adjust their lifestyles and occupations accordingly. 20 refs

  20. Lack of collagen VI promotes neurodegeneration by impairing autophagy and inducing apoptosis during aging.

    Science.gov (United States)

    Cescon, Matilde; Chen, Peiwen; Castagnaro, Silvia; Gregorio, Ilaria; Bonaldo, Paolo

    2016-05-01

    Collagen VI is an extracellular matrix (ECM) protein with a broad distribution in different tissues and mostly deposited at the close periphery of the cell surface. Previous studies revealed that collagen VI protects neurons from the toxicity of amyloid-βpeptides and from UV-induced damage. However, the physiological role of this protein in the central nervous system (CNS) remains unknown. Here, we established primary neural cultures from murine cortex and hippocampus, and carried out in vitro and in vivo studies in wild-type and collagen VI null (Col6a1-/-) mice. Col6a1-/- neural cultures displayed an increased incidence of spontaneous apoptosis and higher vulnerability to oxidative stress, accompanied by altered regulation of autophagy with increased p62 protein levels and decreased LC3 lipidation. Analysis of brain sections confirmed increased apoptosis and abnormal regulation of autophagy in the CNS of collagen VI-deficient animals. To investigate the in vivo physiological consequences of these CNS defects, we carried out functional studies and found that motor and memory task performances were impaired in aged Col6a1-/-mice. These findings indicate that lack of collagen VI leads to spontaneous apoptosis and defective autophagy in neural cells, and point at a protective role for this ECM protein in the CNS during physiological aging. PMID:27060109

  1. Luteolin decreases the UVA‑induced autophagy of human skin fibroblasts by scavenging ROS.

    Science.gov (United States)

    Yan, Miaomiao; Liu, Zhongrong; Yang, Huilan; Li, Cuihua; Chen, Hulin; Liu, Yan; Zhao, Minling; Zhu, Yingjie

    2016-09-01

    Luteolin (LUT) is a flavone, which is universally present as a constituent of traditional Chinese herbs, and certain vegetables and spices, and has been demonstrated to exhibit potent radical scavenging and cytoprotective properties. Although LUT has various beneficial effects on health, the effects of LUT on the protection of skin remain to be fully elucidated. The present study investigated whether LUT can protect human skin fibroblasts (HSFs) from ultraviolet (UV) A irradiation. It was found that, following exposure to different doses of UVA irradiation, the HSFs exhibited autophagy, as observed by fluorescence and transmission electron microscopy, and reactive oxygen species (ROS) bursts, analyzed by flow cytometry, to differing degrees. Following incubation with micromolar concentrations of LUT, ROS production decreased and autophagy gradually declined. In addition, the expression of hypoxia‑inducible factor‑1α and the classical autophagy‑associated proteins, LC3 and Beclin 1 were observed by western blotting. Western blot analysis showed that the expression levels of HIF‑1α, LC3‑II and Beclin 1 gradually decreased in the UVA‑irradiated HSFs following treatment with LUT. These data indicated that UVA‑induced autophagy was mediated by ROS, suggesting the possibility of resistance against UV by certain natural antioxidants, including LUT. PMID:27430964

  2. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

    Directory of Open Access Journals (Sweden)

    Chuanyu Wei

    Full Text Available BACKGROUND: Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage. METHODS: Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated. RESULTS: Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the

  3. The Neuroprotective Effect of Erythropoietin on Rotenone-Induced Neurotoxicity in SH-SY5Y Cells Through the Induction of Autophagy.

    Science.gov (United States)

    Jang, Wooyoung; Kim, Hee Ju; Li, Huan; Jo, Kwang Deog; Lee, Moon Kyu; Yang, Hyun Ok

    2016-08-01

    Currently, the autophagy pathway is thought to be important for the pathogenesis of Parkinson's disease (PD), and the modulation of autophagy may be a novel strategy for the treatment of this disease. Erythropoietin (EPO) has been reported to have neuroprotective effects through anti-oxidative, anti-apoptotic, and anti-inflammatory mechanisms, and it has also been shown to modulate autophagy signaling in an oxygen toxicity model. Therefore, we investigated the effects of EPO on autophagy markers and evaluated its neuroprotective effect on rotenone-induced neurotoxicity. We adapted the rotenone-induced neurotoxicity model to SH-SY5Y cells as an in vitro model of PD. We measured cell viability using MTT and annexin V/propidium iodide assays and measured intracellular levels of reactive oxygen species. Immunofluorescence analysis was performed to measure the expression of LC3 and α-synuclein. Intracellular signaling proteins associated with autophagy were examined by immunoblot analysis. EPO mono-treatment increased the levels of mammalian target of rapamycin (mTOR)-independent/upstream autophagy markers, including Beclin-1, AMPK, and ULK-1. Rotenone treatment of SH-SY5Y cells reduced their viability, increased reactive oxygen species levels, and induced apoptosis and α-synuclein expression, and simultaneous exposure to EPO significantly reduced these effects. Rotenone enhanced mTOR expression and suppressed Beclin-1 expression, indicating suppression of the autophagy system. However, combined treatment with EPO restored Beclin-1 expression and decreased mTOR expression. EPO protects against rotenone-induced neurotoxicity in SH-SY5Y cells by enhancing autophagy-related signaling pathways. The experimental evidence for the EPO-induced neuroprotection against rotenone-induced dopaminergic neurotoxicity may significantly impact the development of future PD treatment strategies. PMID:26156288

  4. Research Progression of Cellular Autophagy in Liver System Diseases

    Directory of Open Access Journals (Sweden)

    Chunyun Liu

    2013-09-01

    Full Text Available Autophagy is a basic biological phenomenon widely existed in eukaryotic cells and an important mechanism for cells to adjust to the surrounding environment, prevent invasion of pathogenic micro-organisms and maintain homeostasis, whose activity changes evidently in multiple liver system diseases, suggesting that there is close association between autophagy and the generation and development of liver system diseases. It is also reported that autophagy develops and exerts an important function in many liver-related diseases, such as hepatic carcinoma, non-alcoholic fatty liver disease, alcoholic liver disease, viral liver disease and acute liver injury. Therefore, this study aimed to summarize the relationship between autophagy and multiple liver diseases, hoping to explore the effect of autophagy in liver system diseases and further study the regulative effect of autophagy so as to provide new thoughts for their treatment.

  5. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth

    DEFF Research Database (Denmark)

    Wild, Philipp; Farhan, Hesso; McEwan, David G; Wagner, Sebastian; Rogov, Vladimir V; Brady, Nathan R; Richter, Benjamin; Korac, Jelena; Waidmann, Oliver; Choudhary, Chunaram; Dötsch, Volker; Bumann, Dirk; Dikic, Ivan

    2011-01-01

    Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms...... controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding...... affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might...

  6. Nanomaterial-modulated autophagy: underlying mechanisms and functional consequences.

    Science.gov (United States)

    Zheng, Wei; Wei, Min; Li, Song; Le, Weidong

    2016-06-01

    Autophagy is an essential lysosome-dependent process that controls the quality of the cytoplasm and maintains cellular homeostasis, and dysfunction of this protein degradation system is correlated with various disorders. A growing body of evidence suggests that nanomaterials (NMs) have autophagy-modulating effects, thus predicting a valuable and promising application potential of NMs in the diagnosis and treatment of autophagy-related diseases. NMs exhibit unique physical, chemical and biofunctional properties, which may endow NMs with capabilities to modulate autophagy via various mechanisms. The present review highlights the impacts of various NMs on autophagy and their functional consequences. The possible underlying mechanisms for NM-modulated autophagy are also discussed. PMID:27193191

  7. Forms, Crosstalks, and the Role of Phospholipid Biosynthesis in Autophagy

    Directory of Open Access Journals (Sweden)

    Leanne Pereira

    2012-01-01

    Full Text Available Autophagy is a highly conserved cellular process occurring during periods of stress to ensure a cell's survival by recycling cytosolic constituents and making products that can be used in energy generation and other essential processes. Three major forms of autophagy exist according to the specific mechanism through which cytoplasmic material is transported to a lysosome. Chaperone-mediated autophagy is a highly selective form of autophagy that delivers specific proteins for lysosomal degradation. Microautophagy is a less selective form of autophagy that occurs through lysosomal membrane invaginations, forming tubes and directly engulfing cytoplasm. Finally, macroautophagy involves formation of new membrane bilayers (autophagosomes that engulf cytosolic material and deliver it to lysosomes. This review provides new insights on the crosstalks between different forms of autophagy and the significance of bilayer-forming phospholipid synthesis in autophagosomal membrane formation.

  8. Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

    Science.gov (United States)

    Stewart, J; Ko, Y-H; Kennedy, A R

    2007-06-01

    Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

  9. Changes in Gene Expression in the Hippocampus Following Exposure to 56Fe Particles and Protection by Berry Diets

    Science.gov (United States)

    Shukitt-Hale, Barbara; Lau, Francis; Carey, Amanda; Carrihill-Knoll, Kirsty; Rabin, Bernard; Joseph, James

    DNA damage caused by radiation to new cells. Supplementation with the berry diets enhanced neuronal communication and cell signaling by altering gene regulation of some of the protective stress signals. Therefore, these data suggest that 56 Fe particle irradiation causes deficits in gene expression in rats which are ameliorated by berry fruit diets.

  10. WASH inhibits autophagy through suppression of Beclin 1 ubiquitination

    OpenAIRE

    Xia, Pengyan; Wang, Shuo; Du, Ying; Zhao, Zhenao; Shi, Lei; Sun, Lei; Huang, Guanling; Ye, Buqing; Li, Chong; Dai, Zhonghua; Hou, Ning; Cheng, Xuan; Sun, Qingyuan; Li, Lei(Beijing Institute of Petrochemical Technology, Beijing, 102617, People's Republic of China); Yang, Xiao

    2013-01-01

    Autophagy degrades cytoplasmic proteins and organelles to recycle cellular components that are required for cell survival and tissue homeostasis. However, it is not clear how autophagy is regulated in mammalian cells. WASH (Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue) plays an essential role in endosomal sorting through facilitating tubule fission via Arp2/3 activation. Here, we demonstrate a novel function of WASH in modulation of autophagy. We show that WASH deficiency causes...

  11. Autophagy and mitophagy in the myocardium: therapeutic potential and concerns

    OpenAIRE

    Jimenez, Rebecca E; Kubli, Dieter A.; Gustafsson, Åsa B.

    2014-01-01

    The autophagic-lysosomal degradation pathway is critical for cardiac homeostasis, and defects in this pathway are associated with development of cardiomyopathy. Autophagy is responsible for the normal turnover of organelles and long-lived proteins. Autophagy is also rapidly up-regulated in response to stress, where it rapidly clears dysfunctional organelles and cytotoxic protein aggregates in the cell. Autophagy is also important in clearing dysfunctional mitochondria before they can cause ha...

  12. Carcinoembryonic Antigen Expression and Resistance to Radiation and 5-Fluorouracil-Induced Apoptosis and Autophagy.

    Science.gov (United States)

    Eftekhar, Ebrahim; Jaberie, Hajar; Naghibalhossaini, Fakhraddin

    2016-01-01

    Understanding the mechanism of tumor resistance is critical for cancer therapy. In this study, we investigated the effect of carcinoembryonic antigen (CEA) overexpression on UV-and 5-fluorouracil (5-FU)-induced apoptosis and autophagy in colorectal cancer cells. We used histone deacetylase (HDAC) inhibitor, NaB and DNA demethylating agent, 5-azacytidine (5-AZA) to induce CEA expression in HT29/219 and SW742 colorectal cancer cell lines. MTT assay was used to measure IC50 value of the cells exposed to graded concentrations of 5- FU with either 0.1 mM NaB or 1 μM 5-AZA for 72 h . Using CHO- and SW742-CEA transfectants, we also investigated the effect of CEA expression on UV- and 5-FU-induced apoptosis and autophagy. Treatment of HT29/219 cell line with NaB and 5-AZA increased CEA expression by 29% and 31%, respectively. Compared with control cells, the IC50 value for 5-FU of NaB and 5-AZA-treated cells increased by 40% and 57%, respectively. Treatment of SW742 cells with NaB or 5-AZA increased neither CEA expression nor the IC50 value for 5-FU. In comparison to parental cells, CEA expression also significantly protected transfected cells against UV-induced apoptosis. Decreased proportions of autophagy and apoptosis were also observed in 5-FU treated SW742- and CHO-CEA transfectants. We conclude that CEA expression can effectively protect colorectal cancer cells against radiation and drug-induced apoptosis and autophagy. PMID:27478804

  13. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    OpenAIRE

    Chen Dishi; Shi Qian; Luo Yan; Yan Qigui; Xu Zhiwen; Guo Wanzhu; Chen Yang; Zhu Ling; Wang Xiaoyu

    2011-01-01

    Abstract Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and...

  14. Autophagy in Lepidoptera: more than old wine in new bottle

    Directory of Open Access Journals (Sweden)

    G Tettamanti

    2011-01-01

    Full Text Available Autophagy is a cellular pathway that leads to the degradation of proteins and organelles. This process is usually involved in the maintenance of cell homeostasis when the organism experiences nutrient starvation, but in holometabolous insects autophagy also intervenes in the demolition of larval tissues and organs during metamorphosis. This review summarizes the current knowledge about autophagy research in Lepidoptera and discusses the use of moths and butterflies as models for tudying the roles and regulation of autophagy. It also gives insights into the cooperation between utophagy and apoptosis in cell death events that occur in lepidopteran in vivo and in vitro systems.

  15. Tracking autophagy during proliferation and differentiation of Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    William R. Proto

    2014-01-01

    Full Text Available Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei.

  16. Autophagy is required for IL-2-mediated fibroblast growth

    International Nuclear Information System (INIS)

    Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts

  17. Autophagy is required for IL-2-mediated fibroblast growth

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Rui [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15219 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15219 (United States); Lotze, Michael T., E-mail: lotzemt@upcm.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15219 (United States); Zeh III, Herbert J., E-mail: zehh@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15219 (United States)

    2013-02-15

    Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts.

  18. Emerging role of selective autophagy in human diseases.

    Directory of Open Access Journals (Sweden)

    Kenji eMizumura

    2014-11-01

    Full Text Available AbstractAutophagy was originally described as a highly conserved system for the degradation of cytosol through a lysosome-dependent pathway. In response to starvation, autophagy degrades organelles and proteins to provide metabolites and energy for its pro-survival effects. Autophagy is recognized as playing a role in the pathogenesis of disease either directly or indirectly, through the regulation of vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms. Recent studies have demonstrated that autophagy is not only a simple metabolite recycling system, but also has the ability to degrade specific cellular targets, such as mitochondria, cilia, and invading bacteria. In addition, selective autophagy has also been implicated in vesicle trafficking pathways, with potential roles in secretion and other intracellular transport processes. Selective autophagy has drawn the attention of researchers because of its potential importance in clinical diseases. Therapeutic strategies to target selective autophagy rather than general autophagy may maximize clinical benefit by enhancing selectivity. In this review, we outline the principle components of selective autophagy processes and their emerging importance in human disease, with an emphasis on pulmonary diseases.

  19. Regulation of autophagy by cytosolic acetyl-coenzyme A

    DEFF Research Database (Denmark)

    Mariño, Guillermo; Pietrocola, Federico; Eisenberg, Tobias;

    2014-01-01

    proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high Ac......CoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a...

  20. Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins.

    Science.gov (United States)

    Manzano-Román, Raúl; Díaz-Martín, Verónica; Oleaga, Ana; Siles-Lucas, Mar; Pérez-Sánchez, Ricardo

    2012-04-30

    Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules. PMID:22105082

  1. A novel contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting autophagy in host cells

    Science.gov (United States)

    Chu, Yuanyuan; Gao, Song; Wang, Ting; Yan, Jing; Xu, Guangmei; Li, Yuanyuan; Niu, Hua; Huang, Rui; Wu, Shuyan

    2016-01-01

    Salmonella plasmid virulence genes (spv) are highly conserved in strains of clinically important Salmonella serovars. It is essential for Salmonella plasmid-correlated virulence, although the exact mechanism remains to be elucidated. Autophagy has been reported to play an important role in host immune responses limiting Salmonella infection. Our previous studies demonstrated that Salmonella conjugative plasmid harboring spv genes could enhance bacterial cytotoxicity by inhibiting autophagy. In the present study, we investigated whether spvB, which is one of the most important constituents of spv ORF could intervene in autophagy pathway. Murine macrophage-like cells J774A.1, human epithelial HeLa cells, and BALB/c mice infected with Salmonella Typhimurium wild type, mutant and complementary strains (carrying or free spvB or complemented only with ADP-ribosyltransferase activity of SpvB) were used in vitro and in vivo assay, respectively. To further explore the molecular mechanisms, both SpvB ectopic eukaryotic expression system and cells deficient in essential autophagy components by siRNA were generated. Results indicated that spvB could suppress autophagosome formation through its function in depolymerizing actin, and aggravate inflammatory injury of the host in response to S. Typhimurium infection. Our studies demonstrated virulence of spvB involving in inhibition of autophagic flux for the first time, which could provide novel insights into Salmonella pathogenesis, and have potential application to develop new antibacterial strategies for Salmonellosis. PMID:26811498

  2. A novel contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting autophagy in host cells.

    Science.gov (United States)

    Chu, Yuanyuan; Gao, Song; Wang, Ting; Yan, Jing; Xu, Guangmei; Li, Yuanyuan; Niu, Hua; Huang, Rui; Wu, Shuyan

    2016-02-16

    Salmonella plasmid virulence genes (spv) are highly conserved in strains of clinically important Salmonella serovars. It is essential for Salmonella plasmid-correlated virulence, although the exact mechanism remains to be elucidated. Autophagy has been reported to play an important role in host immune responses limiting Salmonella infection. Our previous studies demonstrated that Salmonella conjugative plasmid harboring spv genes could enhance bacterial cytotoxicity by inhibiting autophagy. In the present study, we investigated whether spvB, which is one of the most important constituents of spv ORF could intervene in autophagy pathway. Murine macrophage-like cells J774A.1, human epithelial HeLa cells, and BALB/c mice infected with Salmonella Typhimurium wild type, mutant and complementary strains (carrying or free spvB or complemented only with ADP-ribosyltransferase activity of SpvB) were used in vitro and in vivo assay, respectively. To further explore the molecular mechanisms, both SpvB ectopic eukaryotic expression system and cells deficient in essential autophagy components by siRNA were generated. Results indicated that spvB could suppress autophagosome formation through its function in depolymerizing actin, and aggravate inflammatory injury of the host in response to S. Typhimurium infection. Our studies demonstrated virulence of spvB involving in inhibition of autophagic flux for the first time, which could provide novel insights into Salmonella pathogenesis, and have potential application to develop new antibacterial strategies for Salmonellosis. PMID:26811498

  3. Gene and noncoding RNA regulation underlying photoreceptor protection: microarray study of dietary antioxidant saffron and photobiomodulation in rat retina

    OpenAIRE

    Natoli, Riccardo; Zhu, Yuan; Valter, Krisztina; Bisti, Silvia; Eells, Janis; Stone, Jonathan

    2010-01-01

    Purpose To identify the genes and noncoding RNAs (ncRNAs) involved in the neuroprotective actions of a dietary antioxidant (saffron) and of photobiomodulation (PBM). Methods We used a previously published assay of photoreceptor damage, in which albino Sprague Dawley rats raised in dim cyclic illumination (12 h 5 lux, 12 h darkness) were challenged by 24 h exposure to bright (1,000 lux) light. Experimental groups were protected against light damage by pretreatment with dietary saffron (1 mg/kg...

  4. Hexokinase II inhibitor, 3-BrPA induced autophagy by stimulating ROS formation in human breast cancer cells.

    Science.gov (United States)

    Zhang, Qianwen; Zhang, Yuanyuan; Zhang, Pei; Chao, Zhenhua; Xia, Fei; Jiang, Chenchen; Zhang, Xudong; Jiang, Zhiwen; Liu, Hao

    2014-03-01

    Hexokinase II (HKII), a key enzyme of glycolysis, is widely over-expressed in cancer cells. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, has been proposed as a specific antitumor agent. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. Autophagy in mammalian systems occurs under basal conditions and can be stimulated by stresses, including starvation, oxidative stress. Therefore, we hypothesized that 3-BrPA could induce autophagy. In the present study, we explored the mechanism of 3-BrPA and its combined action with chloroquine. Our results demonstrate that in MDA-MB-435 and in MDA-MB-231 cells, 3-BrPA induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment triggered apoptosis in MDA-MB-435 cells, while it induced necroptosis in MDA-MB-231 cells. ROS mediated cell death when 3-BrPA and CQ were co-administered. Finally, CQ enhanced the anticancer efficacy of 3-BrPA in vivo. Collectively, our results show that 3-BrPA triggers autophagy, increasing breast cancer cell resistance to 3-BrPA treatment and that CQ enhanced 3-BrPA-induced cell death in breast cancer cells by stimulating ROS formation. Thus, inhibition of autophagy may be an innovative strategy for adjuvant chemotherapy of breast cancer.human skeletal muscle. Efficient Mirk depletion in SU86.86 pancreatic cancer cells by an inducible shRNA decreased expression of eight antioxidant genes. Thus both cancer cells and differentiated myotubes utilize Mirk kinase to relieve oxidative stress. PMID:25053988

  5. Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

    Science.gov (United States)

    Brueggeman, Andrew J; Bruggeman, Andrew J; Kuehler, Daniel; Weeks, Donald P

    2014-09-01

    Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides. PMID:24796724

  6. Induction of neuro-protective/regenerative genes in stem cells infiltrating post-ischemic brain tissue

    Directory of Open Access Journals (Sweden)

    Yilmaz Gokhan

    2010-05-01

    Full Text Available Abstract Background- Although the therapeutic potential of bone marrow-derived stromal stem cells (BMSC has been demonstrated in different experimental models of ischemic stroke, it remains unclear how stem cells (SC induce neuroprotection following stroke. In this study, we describe a novel method for isolating BMSC that infiltrate postischemic brain tissue and use this method to identify the genes that are persistently activated or depressed in BMSC that infiltrate brain tissue following ischemic stroke. Methods- Ischemic strokes were induced in C57BL/6 mice by middle cerebral artery occlusion for 1 h, followed by reperfusion. BMSC were isolated from H-2 Kb-tsA58 (immortomouse™ mice, and were administered (i.v. 24 h after reperfusion. At the peak of therapeutic improvement (14 days after the ischemic insult, infarcted brain tissue was isolated, and the BMSC were isolated by culturing at 33°C. Microarray analysis and RT-PCR were performed to compare differential gene expression between naïve and infiltrating BMSC populations. Results- Z-scoring revealed dramatic differences in the expression of extracellular genes between naïve and infiltrating BMSC. Pair-wise analysis detected 80 extracellular factor genes that were up-regulated (≥ 2 fold, P Conclusions- BMSC infiltrating the post-ischemic brain exhibit persistent epigenetic changes in gene expression for numerous extracellular genes, compared to their naïve counterparts. These genes are relevant to the neuroprotection, regeneration and angiogenesis previously described following stem cell therapy in animal models of ischemic stroke.

  7. Protective effects of pLNCX-SOD gene transfection on hepatocyte injury induced by obstructive jaundice in rats

    Institute of Scientific and Technical Information of China (English)

    Peng Gong; Zhong-Yu Wang; Hong-Jiang Wang

    2007-01-01

    BACKGROUND: Hydrophobic bile acids lead to the generation of oxygen free radicals in mitochondria. Accordingly, this study is to investigate if gene delivery of superoxide dismutase (SOD) will reduce hepatocyte injury caused by experimental cholestasis. METHODS:The recombinant of pLNCX-SOD gene packaged with lipofection of AMINE was transfected into hepatocytes in vitro, which stably expressed the SOD gene. RESULTS:After transfection, hepatocytes enhanced the protective effect against injury to bile and the toxicity of serum in obstructive jaundice. The inhibition of bile at the concentration of 2% (v∶v, bile: DMEM 1∶50) decreased obviously from (78.80±12.35)% to (43.35±9.69)% in 12 hours, from (82.55±11.27)% to (-26.64±7.66)% in 24 hours, and from (83.83±18.69)% to (-19.27±14.38)% in 48 hours, compared with that of the untransfected cells (P CONCLUSION:Hepatocytes transfected with the pLNCX-SOD gene could obviously be resistant to the toxicity of bile and serum from rat with obstructive jaundice.

  8. Carnosine as a protective factor in diabetic nephropathy: association with a leucine repeat of the carnosinase gene CNDP1.

    Science.gov (United States)

    Janssen, Bart; Hohenadel, Daniela; Brinkkoetter, Paul; Peters, Verena; Rind, Nina; Fischer, Christine; Rychlik, Ivan; Cerna, Marie; Romzova, Marianna; de Heer, Emile; Baelde, Hans; Bakker, Stephan J L; Zirie, Mahmoud; Rondeau, Eric; Mathieson, Peter; Saleem, Moin A; Meyer, Jochen; Köppel, Hannes; Sauerhoefer, Sibylle; Bartram, Claus R; Nawroth, Peter; Hammes, Hans-Peter; Yard, Benito A; Zschocke, Johannes; van der Woude, Fokko J

    2005-08-01

    The risk of diabetic nephropathy is partially genetically determined. Diabetic nephropathy is linked to a gene locus on chromosome 18q22.3-q23. We aimed to identify the causative gene on chromosome 18 and to study the mechanism by which the product of this gene could be involved in the development of diabetic nephropathy. DNA polymorphisms were determined in 135 case (diabetic nephropathy) and 107 control (diabetes without nephropathy) subjects. The effect of carnosine on the production of extracellular matrix components and transforming growth factor-beta (TGF-beta) after exposure to 5 and 25 mmol/l d-glucose was studied in cultured human podocytes and mesangial cells, respectively. A trinucleotide repeat in exon 2 of the CNDP1 gene, coding for a leucine repeat in the leader peptide of the carnosinase-1 precursor, was associated with nephropathy. The shortest allelic form (CNDP1 Mannheim) was more common in the absence of nephropathy (P = 0.0028, odds ratio 2.56 [95% CI 1.36-4.84]) and was associated with lower serum carnosinase levels. Carnosine inhibited the increased production of fibronectin and collagen type VI in podocytes and the increased production of TGF-beta in mesangial cells induced by 25 mmol/l glucose. Diabetic patients with the CNDP1 Mannheim variant are less susceptible for nephropathy. Carnosine protects against the adverse effects of high glucose levels on renal cells. PMID:16046297

  9. Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specific phototoxic molecules.

    OpenAIRE

    Tuveson, R W; Larson, R A; Kagan, J.

    1988-01-01

    Genes controlling carotenoid synthesis were cloned from Erwinia herbicola and expressed in an Escherichia coli strain. Carotenoids protect against high fluences of near-UV (NUV; 320 to 400 nm) but not against far-UV (200-300 nm). Protection of E. coli cells was not observed following treatment with either psoralen or 8-methoxypsoralen plus NUV. However, significant protection of cells producing carotenoids was observed with three photosensitizing molecules activated by NUV (alpha-terthienyl, ...

  10. Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease.

    Science.gov (United States)

    Chu, Hiutung; Khosravi, Arya; Kusumawardhani, Indah P; Kwon, Alice H K; Vasconcelos, Anilton C; Cunha, Larissa D; Mayer, Anne E; Shen, Yue; Wu, Wei-Li; Kambal, Amal; Targan, Stephan R; Xavier, Ramnik J; Ernst, Peter B; Green, Douglas R; McGovern, Dermot P B; Virgin, Herbert W; Mazmanian, Sarkis K

    2016-05-27

    Inflammatory bowel disease (IBD) is associated with risk variants in the human genome and dysbiosis of the gut microbiome, though unifying principles for these findings remain largely undescribed. The human commensal Bacteroides fragilis delivers immunomodulatory molecules to immune cells via secretion of outer membrane vesicles (OMVs). We reveal that OMVs require IBD-associated genes, ATG16L1 and NOD2, to activate a noncanonical autophagy pathway during protection from colitis. ATG16L1-deficient dendritic cells do not induce regulatory T cells (T(regs)) to suppress mucosal inflammation. Immune cells from human subjects with a major risk variant in ATG16L1 are defective in T(reg) responses to OMVs. We propose that polymorphisms in susceptibility genes promote disease through defects in "sensing" protective signals from the microbiome, defining a potentially critical gene-environment etiology for IBD. PMID:27230380

  11. Grouping annotations on the subcellular layered interactome demonstrates enhanced autophagy activity in a recurrent experimental autoimmune uveitis T cell line.

    Directory of Open Access Journals (Sweden)

    Xiuzhi Jia

    Full Text Available Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse-remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652 between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.

  12. Autophagy sensitivity of neuroendocrine lung tumor cells

    OpenAIRE

    HONG, SEUNG-KEUN; Kim, Jin-Hwan; Starenki, Dmytro; Park, Jong-In

    2013-01-01

    Neuroendocrine (NE) phenotypes characterize a spectrum of lung tumors, including low-grade typical and intermediate-grade atypical carcinoid, high-grade large-cell NE carcinoma and small cell lung carcinoma. Currently, no effective treatments are available to cure NE lung tumors, demanding identification of biological features specific to these tumors. Here, we report that autophagy has an important role for NE lung tumor cell proliferation and survival. We found that the expression levels of...

  13. Cardiomyocyte autophagy: metabolic profit and loss

    OpenAIRE

    Wang, Zhao V.; Ferdous, Anwarul; Hill, Joseph A.

    2013-01-01

    Cardiovascular disease remains the leading cause of morbidity and mortality worldwide, even despite recent scientific and technological advances and comprehensive preventive strategies. The cardiac myocyte is a voracious consumer of energy, and alterations in metabolic substrate availability and consumption are hallmark features of these disorders. Autophagy, an evolutionarily ancient response to metabolic insufficiency, has been implicated in the pathogenesis of a wide range of heart patholo...

  14. Autophagy in Mycobacterium tuberculosis and HIV infections

    OpenAIRE

    Espert, Lucile; Beaumelle, Bruno; Vergne, Isabelle

    2015-01-01

    Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis (M.tb) are among the most lethal human pathogens worldwide, each being responsible for around 1.5 million deaths annually. Moreover, synergy between acquired immune deficiency syndrome (AIDS) and tuberculosis (TB) has turned HIV/M.tb co-infection into a major public health threat in developing countries. In the past decade, autophagy, a lysosomal catabolic process, has emerged as a major host immune defense mechanism against in...

  15. The Role of Autophagy in Lupus Nephritis

    OpenAIRE

    Linlin Wang; Helen Ka Wai Law

    2015-01-01

    Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by the generation of immune responses to self-antigens. Lupus nephritis is one of the most common and severe complications in SLE patients. Though the pathogenesis of lupus nephritis has been studied extensively, unresolved questions are still left and new therapeutic methods are needed for disease control. Autophagy is a conserved catabolic process through which cytoplasmic constituents can be degraded in...

  16. Autophagy extends lifespan via vacuolar acidification

    Directory of Open Access Journals (Sweden)

    Christoph Ruckenstuhl

    2014-05-01

    Full Text Available Methionine restriction (MetR is one of the rare regimes that prolongs lifespan across species barriers. Using a yeast model, we recently demonstrated that this lifespan extension is promoted by autophagy, which in turn requires vacuolar acidification. Our study is the first to place autophagy as one of the major players required for MetR-mediated longevity. In addition, our work identifies vacuolar acidification as a key downstream element of autophagy induction under MetR, and possibly after rapamycin treatment. Unlike other amino acids, methionine plays pleiotropic roles in many metabolism-relevant pathways. For instance, methionine (i is the N-terminal amino acid of every newly translated protein; (ii acts as the central donor of methyl groups through S-adenosyl methionine (SAM during methylation reactions of proteins, DNA or RNA; and (iii provides the sulfhydryl groups for FeS-cluster formation and redox detoxification via transsulfuration to cysteine. Intriguingly, MetR causes lifespan extension, both in yeast and in rodents. We could show that in Saccharomyces cerevisiae, chronological lifespan (CLS is increased in two specific methionine-auxotrophic strains (namely Δmet2 and Δmet15.

  17. Gene expression profiling reveals novel protective effects of Aminaphtone on ECV304 endothelial cells.

    Science.gov (United States)

    Salazar, Giulia; Bellocchi, Chiara; Todoerti, Katia; Saporiti, Federica; Piacentini, Luca; Scorza, Raffaella; Colombo, Gualtiero I

    2016-07-01

    Aminaphtone, a drug used in the treatment of chronic venous insufficiency (CVI), showed a remarkable role in the modulation of several vasoactive factors, like endothelin-1 and adhesion molecules. We analysed in vitro the effects of Aminaphtone on whole-genome gene expression and production of different inflammatory proteins. ECV-304 endothelial cells were stimulated with IL-1β 100U/ml in the presence or absence of Aminaphtone 6μg/ml. Gene expression profiles were compared at 1, 3, and 6h after stimulation by microarray. Supernatants of ECV-304 cultures were analysed at 3, 6, 12, and 24h by multiplex ELISA for production of several cytokine and chemokines. Microarrays showed a significant down-regulation at all times of a wide range of inflammatory genes. Aminaphtone appeared also able to modulate the regulation of immune response process (down-regulating cytokine biosynthesis, transcripts involved in lymphocyte differentiation and cell proliferation, and cytokine-cytokine receptor interaction) and to regulate genes engaged in homeostasis, secretion, body fluid levels, response to hypoxia, cell division, and cell-to-cell communication and signalling. Results were confirmed and extended analysing the secretome, which showed significant reduction of the release of 14 cytokines and chemokines. These effects are predicted to be mediated by interaction with different transcription factors. Aminaphtone was able to modulate the expression of inflammatory molecules relevant to the pathogenesis of several conditions in which the endothelial dysfunction is the main player and early event, like scleroderma, lung fibrosis, or atherosclerosis. PMID:27083548

  18. A Cyanobacterium Synechocystis sp. PCC 6803 Glutaredoxin Gene (slr1562 Protects Escherichia coli against Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    Ahmed Gaber

    2012-01-01

    Full Text Available Problem statement: Glutaredoxins (GRXs are ubiquitous small heat stable glutathione-dependent oxidoreductase enzymes that play a crucial role in plant development and response to oxidative stress. Approach: Cyanobacterium Synechocystis strain PCC 6803 contains two genes (slr1562 and ssr2061 encoding glutaredoxins. In the present investigation the slr1562 gene (grxC was isolated and characterized. Results: The results revealed that the amino acid sequence deduced from GrxC protein share high identity with those of GRXs from other organisms and contain the consensus GRX family domain with a CPFC active site. Northern blotting analysis revealed that the expression of slr1562 gene could be induced by oxidative and salt stresses. Moreover, the protein GrxC was successfully overexpressed as a soluble fraction in Escherichia coli JM109. The over-expression of GrxC in Escherichia coli cells significantly increased resistance of cells to oxidative, drought and salt stresses. Conclusion/Recommendations: These results suggest that the slr1562 gene could play an important role in regulating abiotic tolerance against oxidative, drought and salt stresses in different organisms.

  19. Moderate exercise training provides modest protection against adipose tissue inflammatory gene expression in response to high-fat feeding.

    Science.gov (United States)

    Linden, Melissa A; Pincu, Yair; Martin, Stephen A; Woods, Jeffrey A; Baynard, Tracy

    2014-01-01

    As white adipose tissue (WAT) expands under obesogenic conditions, local WAT hypoxia may contribute to the chronic low-grade inflammation observed in obesity. Aerobic exercise training is beneficial in treating WAT inflammation after obesity is established, but it remains unknown whether exercise training, while on a concomitant high-fat (HF) diet, influences WAT inflammation during the development of obesity. We sought to determine the effects of 4, 8, and 12 weeks of HF feeding and/or moderate intensity treadmill exercise training (EX) on the relationship between inflammatory and hypoxic gene expression within mouse WAT. Male C57Bl6/J mice (n = 113) were randomized into low-fat (LF)/sedentary (SED), LF/EX, HF/SED, or HF/EX groups. The low-fat and high-fat diets contained 10% and 60% energy from fat, respectively. Exercise training consisted of treadmill running 5 days/week at 12 m/min, 8% incline, 40 min/day. Quantitative real-time PCR was used to assess gene expression. HF diet impaired glucose regulation, and upregulated WAT gene expression of inflammation (IL-1β, IL-1ra, TNFα), macrophage recruitment and infiltration (F4/80 and monocyte chemoattractant protein), and M1 (CD11c) and M2 (CD206 and Arginase-1) macrophage polarization markers. Treadmill training resulted in a modest reduction of WAT macrophage and inflammatory gene expression. HF diet had little effect on hypoxia-inducible factor-1α and vascular endothelial growth factor, suggesting that WAT inflammatory gene expression may not be driven by hypoxia within the adipocytes. Treadmill training may provide protection by preventing WAT expansion and macrophage recruitment. PMID:25347855

  20. Autophagy Proteins ATG5 and ATG7 Are Essential for the Maintenance of Human CD34(+) Hematopoietic Stem-Progenitor Cells.

    Science.gov (United States)

    Gomez-Puerto, Maria Catalina; Folkerts, Hendrik; Wierenga, Albertus T J; Schepers, Koen; Schuringa, Jan Jacob; Coffer, Paul J; Vellenga, Edo

    2016-06-01

    Autophagy is a highly regulated catabolic process that involves sequestration and lysosomal degradation of cytosolic components such as damaged organelles and misfolded proteins. While autophagy can be considered to be a general cellular housekeeping process, it has become clear that it may also play cell type-dependent functional roles. In this study, we analyzed the functional importance of autophagy in human hematopoietic stem/progenitor cells (HSPCs), and how this is regulated during differentiation. Western blot-based analysis of LC3-II and p62 levels, as well as flow cytometry-based autophagic vesicle quantification, demonstrated that umbilical cord blood-derived CD34(+) /CD38(-) immature hematopoietic progenitors show a higher autophagic flux than CD34(+) /CD38(+) progenitors and more differentiated myeloid and erythroid cells. This high autophagic flux was critical for maintaining stem and progenitor function since knockdown of autophagy genes ATG5 or ATG7 resulted in reduced HSPC frequencies in vitro as well as in vivo. The reduction in HSPCs was not due to impaired differentiation, but at least in part due to reduced cell cycle progression and increased apoptosis. This is accompanied by increased expression of p53, proapoptotic genes BAX and PUMA, and the cell cycle inhibitor p21, as well as increased levels of cleaved caspase-3 and reactive oxygen species. Taken together, our data demonstrate that autophagy is an important regulatory mechanism for human HSCs and their progeny, reducing cellular stress and promoting survival. Stem Cells 2016;34:1651-1663. PMID:26930546

  1. Salmonella enterica Serovar Typhimurium Lacking hfq Gene Confers Protective Immunity against Murine Typhoid

    OpenAIRE

    Allam, Uday Shankar; Krishna, Gopala M; Lahiri, Amit; Joy, Omana; Chakravortty, Dipshikha

    2011-01-01

    Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generat...

  2. The lipoprotein lipase gene in combined hyperlipidemia: evidence of a protective allele depletion

    OpenAIRE

    Malloy Mary J; Pullinger Clive R; Kulkarni Medha V; Wung Shu-Fen; Kane John P; Aouizerat Bradley E

    2006-01-01

    Abstract Background Lipoprotein Lipase (LPL), a key enzyme in lipid metabolism, catalyzes the hydrolysis of triglycerides (TG) from TG-rich lipoproteins, and serves a bridging function that enhances the cellular uptake of lipoproteins. Abnormalities in LPL function are associated with pathophysiological conditions, including familial combined hyperlipidemia (FCH). Whereas two LPL susceptibility alleles were found to co-segregate in a few FCH kindred, a role for common, protective alleles rema...

  3. Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline.

    Science.gov (United States)

    Schwarz, Flavio; Springer, Stevan A; Altheide, Tasha K; Varki, Nissi M; Gagneux, Pascal; Varki, Ajit

    2016-01-01

    The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer's dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer's disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin. PMID:26621708

  4. Cardiac-specific catalase overexpression rescues anthrax lethal toxin-induced cardiac contractile dysfunction: role of oxidative stress and autophagy

    Directory of Open Access Journals (Sweden)

    Kandadi Machender R

    2012-11-01

    Full Text Available Abstract Background Lethal and edema toxins secreted by Bacillus anthracis during anthrax infection were found to incite serious cardiovascular complications. However, the underlying mechanisms in anthrax lethal toxin-induced cardiac anomalies remain unknown. This study was designed to evaluate the impact of antioxidant enzyme catalase in anthrax lethal toxin-induced cardiomyocyte contractile dysfunction. Methods Wild type (WT and cardiac-specific catalase overexpression mice were challenged with lethal toxin (2 μg/g, intraperotineally (i.p.. Cardiomyocyte contractile and intracellular Ca2+ properties were assessed 18 h later using an IonOptix edge-detection system. Proteasome function was assessed using chymotrypsin-like and caspase-like activities. GFP-LC3 puncta and Western blot analysis were used to evaluate autophagy and protein ubiquitination. Results Lethal toxin exposure suppressed cardiomyocyte contractile function (suppressed peak shortening, maximal velocity of shortening/re-lengthening, prolonged duration of shortening/re-lengthening, and impaired intracellular Ca2+ handling, the effects of which were alleviated by catalase. In addition, lethal toxin triggered autophagy, mitochondrial and ubiquitin-proteasome defects, the effects of which were mitigated by catalase. Pretreatment of cardiomyocytes from catalase mice with the autophagy inducer rapamycin significantly attenuated or ablated catalase-offered protection against lethal toxin-induced cardiomyocyte dysfunction. On the other hand, the autophagy inhibitor 3-MA ablated or significantly attenuated lethal toxin-induced cardiomyocyte contractile anomalies. Conclusions Our results suggest that catalase is protective against anthrax lethal toxin-induced cardiomyocyte contractile and intracellular Ca2+ anomalies, possibly through regulation of autophagy and mitochondrial function.

  5. Puerarin Attenuates Anoxia/Reoxygenation Injury Through Enhancing Bcl-2 Associated Athanogene 3 Expression, a Modulator of Apoptosis and Autophagy.

    Science.gov (United States)

    Ma, Yayu; Gai, Ya; Yan, Jingpeng; Li, Jian; Zhang, Yangyang

    2016-01-01

    BACKGROUND Puerarin has protective effects on ischemia-reperfusion injury, but the underlying mechanisms are not fully revealed. This study explored the effect of puerarin on the expression of Bcl-2 associated athanogene 3 (BAG3) in an in vitro model of anoxia/reoxygenation injury (A/RI) in neonate rat primary cardiomyocytes and the functions of BAG3 in A/RI. MATERIAL AND METHODS BAG3 expression in cardiomyocytes with or without puerarin pre-treatment was quantified using qRT-PCR and Western blot analysis. The effects of BAG3 on A/RI were studied by measuring the activity of lactate dehydrogenase (LDH) and creatine phosphate kinase (CPK), the concentration of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). The effects of BAG3 on autophagy and apoptosis of the cardiomyocytes after A/RI were further studied. RESULTS Puerarin significantly promoted BAG3 expression in the rat primary cardiomyocytes after A/RI. Enforced BAG3 expression presented similar effects as puerarin pre-treatment in attenuating A/RI in terms of CPK, LDH, MDA, SOD, GSH-Px, ROS generation, and cell viability. BAG3 overexpression significantly stimulated autophagy in cardiomyocytes after A/RI, which presented protective effects on A/RI in terms of cell viability and apoptosis. Autophagy inhibition partly abrogated the protective effects of BAG3. CONCLUSIONS Puerarin can directly increase BAG3 transcription and translation in cardiomyocytes after A/RI. The elevated BAG3 expression presents protective effects on A/RI at least through enhancing autophagy and reducing apoptosis, which is a novel protective mechanism of puerarin in ARI. PMID:27011313

  6. Safety assessment of lepidopteran insect-protected transgenic rice with cry2A* gene.

    Science.gov (United States)

    Zou, Shiying; Huang, Kunlun; Xu, Wentao; Luo, Yunbo; He, Xiaoyun

    2016-04-01

    Numerous genetically modified (GM) crops expressing proteins for insect resistance have been commercialized following extensive testing demonstrating that the foods obtained from them are as safe as that obtained from their corresponding non-GM varieties. In this paper, we report the outcome of safety studies conducted on a newly developed insect-resistant GM rice expressing the cry2A* gene by a subchronic oral toxicity study on rats. GM rice and non-GM rice were incorporated into the diet at levels of 30, 50, and 70% (w/w), No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. These results demonstrate that the GM rice with cry2A* gene is as safe for food as conventional non-GM rice. PMID:26581349

  7. Estrogen-related receptor α is essential for the expression of antioxidant protection genes and mitochondrial function

    International Nuclear Information System (INIS)

    Estrogen-related receptor α (ERRα) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERRα null mice. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) stimulated mitochondrial gene expression program in control cells, but not in the ERRα null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1α levels was dependent on ERRα. Furthermore, we found that the PGC-1α-mediated induction of estrogen-related receptor γ and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERRα. Basal levels of NRF-2 were decreased in the absence of ERRα. The absence of ERRα resulted in a decrease in citrate synthase enzyme activity in response to PGC-1α overexpression. Our results indicate an essential role for ERRα as a key regulator of oxidative metabolism

  8. Altered autophagy in human adipose tissues in obesity

    Science.gov (United States)

    Context: Autophagy is a housekeeping mechanism, involved in metabolic regulation and stress response, shown recently to regulate lipid droplets biogenesis/breakdown and adipose tissue phenotype. Objective: We hypothesized that in human obesity autophagy may be altered in adipose tissue in a fat d...

  9. Autophagy: A double-edged sword in Alzheimer's disease

    Indian Academy of Sciences (India)

    Ying-Tsen Tung; Bo-Jeng Wang; Ming-Kuan Hu; Wen-Ming Hsu; Hsinyu Lee; Wei-Pang Huang; Yung-Feng Liao

    2012-03-01

    Autophagy is a major protein degradation pathway that is essential for stress-induced and constitutive protein turnover. Accumulated evidence has demonstrated that amyloid- (A) protein can be generated in autophagic vacuoles, promoting its extracellular deposition in neuritic plaques as the pathological hallmark of Alzheimer’s disease (AD). The molecular machinery for A generation, including APP, APP-C99 and -/-secretases, are all enriched in autophagic vacuoles. The induction of autophagy can be vividly observed in the brain at early stages of sporadic AD and in an AD transgenic mouse model. Accumulated evidence has also demonstrated a neuroprotective role of autophagy in mediating the degradation of aggregated proteins that are causative of various neurodegenerative diseases. Autophagy is thus widely regarded as an intracellular hub for the removal of the detrimental A peptides and Tau aggregates. Nonetheless, compelling data also reveal an unfavorable function of autophagy in facilitating the production of intracellular A. The two faces of autophagy on the homeostasis of A place it in a very unique and intriguing position in ADpathogenesis. This article briefly summarizes seminal discoveries that are shedding new light on the critical and unique roles of autophagy in AD and potential therapeutic approaches against autophagy-elicited AD.

  10. Autophagy in ageing and ageing-associated diseases

    Institute of Scientific and Technical Information of China (English)

    Li-qiang HE; Jia-hong LU; Zhen-yu YUE

    2013-01-01

    Autophagy is a cell self-digestion process via lysosomes that clears "cellular waste",including aberrantly modified proteins or protein aggregates and damaged organelles.Therefore,autophagy is considered a protein and organelle quality control mechanism that maintains normal cellular homeostasis.Dysfunctional autophagy has been observed in ageing tissues and several ageing-associated diseases.Lifespan of model organisms such as yeast,worms,flies,and mice can be extended through promoting autophagy,either by genetic manipulations such as over-expression of Sirtuin 1,or by administrations of rapamycin,resveratrol or spermidine.The evidence supports that autophagy may play an important role in delaying ageing or extending lifespan.In this review,we summarize the current knowledge about autophagy and its regulation,outline recent developments ie the genetic and pharmacological manipulations of autophagy that affects the lifespan,and discuss the role of autophagy in the ageing-related diseases.ow in Center for Neurodegenerative and Neuroimmunologic Diseases,Department of Neurology,University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School,Piscataway,NJ 08854,USA

  11. Autophagy Inhibition Dysregulates TBK1 Signaling and Promotes Pancreatic Inflammation.

    Science.gov (United States)

    Yang, Shenghong; Imamura, Yu; Jenkins, Russell W; Cañadas, Israel; Kitajima, Shunsuke; Aref, Amir; Brannon, Arthur; Oki, Eiji; Castoreno, Adam; Zhu, Zehua; Thai, Tran; Reibel, Jacob; Qian, Zhirong; Ogino, Shuji; Wong, Kwok K; Baba, Hideo; Kimmelman, Alec C; Pasca Di Magliano, Marina; Barbie, David A

    2016-06-01

    Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK)-related kinase TBK1 in vivo, associated with increased neutrophil and T-cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKε/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting. Cancer Immunol Res; 4(6); 520-30. ©2016 AACR. PMID:27068336

  12. Autophagy: A Potential Link between Obesity and Insulin Resistance

    NARCIS (Netherlands)

    P. Codogno; A.J. Meijer

    2010-01-01

    Dysregulation of autophagy contributes to aging and to diseases such as neurodegeneration, cardiomyopathy, and cancer. The paper by Yang et al. (2010) in this issue of Cell Metabolism indicates that defective autophagy may also underlie impaired insulin sensitivity in obesity and that upregulating a

  13. Protective effect of myostatin gene deletion on aging-related muscle metabolic decline

    OpenAIRE

    Chabi, Beatrice; Pauly, Marion; Carillon, Julie; Carnac, Gilles; Favier, François; Fouret, Gilles; Bonafos, Béatrice; Vanterpool, Frankie; Vernus, Barbara,; Coudray, Charles; Feillet Coudray, Christine; Bonnieu, Anne; Lacan, Dominique

    2016-01-01

    While myostatin gene deletion is a promising therapy to fight muscle loss during aging, this approach induces also skeletal muscle metabolic changes such as mitochondrial deficits, redox alteration and increased fatigability. In the present study, we evaluated the effects of aging on these features in aged wild-type (WT) and mstn knockout (KO) mice. Moreover, to determine whether an enriched-antioxidant diet may be useful to prevent agerelated disorders, we orally administered to the...

  14. Autophagy in RAW264.7 Cells Treated with Surface-Functionalized Graphene Oxides

    Directory of Open Access Journals (Sweden)

    Chang Seok Park

    2015-01-01

    Full Text Available This study investigated cytotoxicity, particularly autophagy, in RAW264.7 cells exposed to graphene oxide (GO and its derivatives (dodecylamine-GO (DA-GO, reduced GO (rGO, and sodium dodecyl sulfate-rGO (SDS-rGO. Appearance of amine stretching bands, out-of-plane C-H stretching vibrations, and S=O stretching bands in infrared spectra indicated the formation of DA-GO, rGO, and SDS-rGO, respectively. Light microscopy and microculture tetrazolium assay showed that all the GO types exerted cytotoxic effects on RAW264.7 cells in a concentration-dependent manner. Higher concentrations of the GO types downregulated the expression of PU.1, a unique transcription factor in monocytes and macrophages, and decreased the conversion of LC3A/B-I to LC3A/B-II, suggesting that PU.1 was associated with autophagy in RAW264.7 cells. These results suggested that surface-functionalized GOs exerted cytotoxic effects in a concentration-dependent manner by changing the expression of critical genes and inducing autophagy in macrophages.

  15. Atorvastatin activates autophagy and promotes neurological function recovery after spinal cord injury

    Science.gov (United States)

    Gao, Shuang; Zhang, Zhong-ming; Shen, Zhao-liang; Gao, Kai; Chang, Liang; Guo, Yue; Li, Zhuo; Wang, Wei; Wang, Ai-mei

    2016-01-01

    Atorvastatin, a lipid-lowering medication, provides neuroprotective effects, although the precise mechanisms of action remain unclear. Our previous studies confirmed activated autophagy following spinal cord injury, which was conducive to recovery of neurological functions. We hypothesized that atorvastatin could also activate autophagy after spinal cord injury, and subsequently improve recovery of neurological functions. A rat model of spinal cord injury was established based on the Allen method. Atorvastatin (5 mg/kg) was intraperitoneally injected at 1 and 2 days after spinal cord injury. At 7 days post-injury, western blot assay, reverse transcription-polymerase chain reaction, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining results showed increased Beclin-1 and light chain 3B gene and protein expressions in the spinal cord injury + atorvastatin group. Additionally, caspase-9 and caspase-3 expression was decreased, and the number of TUNEL-positive cells was reduced. Compared with the spinal cord injury + saline group, Basso, Beattie, and Bresnahan locomotor rating scale scores significantly increased in the spinal cord injury + atorvastatin group at 14–42 days post-injury. These findings suggest that atorvastatin activated autophagy after spinal cord injury, inhibited apoptosis, and promoted recovery of neurological function.

  16. Astrocytes Upregulate Survival Genes in Tumor Cells and Induce Protection from Chemotherapy

    Directory of Open Access Journals (Sweden)

    Sun-Jin Kim

    2011-03-01

    Full Text Available In the United States, more than 40% of cancer patients develop brain metastasis. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy. The growth and survival of metastasis depend on the interaction of tumor cells with host factors in the organ microenvironment. Brain metastases are surrounded and infiltrated by activated astrocytes and are highly resistant to chemotherapy. We report here that coculture of human breast cancer cells or lung cancer cells with murine astrocytes (but not murine fibroblasts led to the up-regulation of survival genes, including GSTA5, BCL2L1, and TWIST1, in the tumor cells. The degree of up-regulation directly correlated with increased resistance to all tested chemotherapeutic agents. We further show that the up-regulation of the survival genes and consequent resistance are dependent on the direct contact between the astrocytes and tumor cells through gap junctions and are therefore transient. Knocking down these genes with specific small interfering RNA rendered the tumor cells sensitive to chemotherapeutic agents. These data clearly demonstrate that host cells in the microenvironment influence the biologic behavior of tumor cells and reinforce the contention that the organ microenvironment must be taken into consideration during the design of therapy.

  17. Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction

    Directory of Open Access Journals (Sweden)

    Zhan-ping He

    2015-01-01

    Full Text Available Aquaporin-4 regulates water molecule channels and is important in tissue regulation and water transportation in the brain. Upregulation of aquaporin-4 expression is closely related to cellular edema after early cerebral infarction. Cellular edema and aquaporin-4 expression can be determined by measuring cerebral infarct area and apparent diffusion coefficient using diffusion-weighted imaging (DWI. We examined the effects of silencing aquaporin-4 on cerebral infarction. Rat models of cerebral infarction were established by occlusion of the right middle cerebral artery and siRNA-aquaporin-4 was immediately injected via the right basal ganglia. In control animals, the area of high signal intensity and relative apparent diffusion coefficient value on T2-weighted imaging (T2WI and DWI gradually increased within 0.5-6 hours after cerebral infarction. After aquaporin-4 gene silencing, the area of high signal intensity on T2WI and DWI reduced, relative apparent diffusion coefficient value was increased, and cellular edema was obviously alleviated. At 6 hours after cerebral infarction, the apparent diffusion coefficient value was similar between treatment and model groups, but angioedema was still obvious in the treatment group. These results indicate that aquaporin-4 gene silencing can effectively relieve cellular edema after early cerebral infarction; and when conducted accurately and on time, the diffusion coefficient value and the area of high signal intensity on T2WI and DWI can reflect therapeutic effects of aquaporin-4 gene silencing on cellular edema.

  18. Cancer-predisposition gene KLLN maintains pericentric H3K9 trimethylation protecting genomic stability.

    Science.gov (United States)

    Nizialek, Emily A; Sankunny, Madhav; Niazi, Farshad; Eng, Charis

    2016-05-01

    Maintenance of proper chromatin states and genomic stability is vital for normal development and health across a range of organisms. Here, we report on the role of KLLN in maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN, a gene uncovered well after the human genome project, has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN wild-type cases. KLLN first identified as a p53-dependent tumor suppressor gene, was believed to bind randomly to DNA and cause S-phase arrest. Using chromatin immunoprecipitation-based sequencing (ChIP-seq), we demonstrated that KLLN binds to DNA regions enriched with H3K9me3. KLLN overexpression correlated with increased H3K9 methyltransferase activity and increased global H3K9me3, while knockdown of KLLN had an opposite effect. We also found KLLN to localize to pericentric regions, with loss of KLLN resulting in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei formation and numerical chromosomal aberrations. Interestingly, we show that KLLN interacts with DBC1, with consequent abrogation of DBC1 inhibition of SUV39H1, a H3K9 methyltransferase, suggesting the mode of KLLN regulating H3K9me3. These results suggest a critical role for KLLN as a potential regulator of pericentric heterochromatin formation, genomic stability and gene expression. PMID:26673699

  19. Cancer-predisposition gene KLLN maintains pericentric H3K9 trimethylation protecting genomic stability

    Science.gov (United States)

    Nizialek, Emily A.; Sankunny, Madhav; Niazi, Farshad; Eng, Charis

    2016-01-01

    Maintenance of proper chromatin states and genomic stability is vital for normal development and health across a range of organisms. Here, we report on the role of KLLN in maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN, a gene uncovered well after the human genome project, has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN wild-type cases. KLLN first identified as a p53-dependent tumor suppressor gene, was believed to bind randomly to DNA and cause S-phase arrest. Using chromatin immunoprecipitation-based sequencing (ChIP-seq), we demonstrated that KLLN binds to DNA regions enriched with H3K9me3. KLLN overexpression correlated with increased H3K9 methyltransferase activity and increased global H3K9me3, while knockdown of KLLN had an opposite effect. We also found KLLN to localize to pericentric regions, with loss of KLLN resulting in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei formation and numerical chromosomal aberrations. Interestingly, we show that KLLN interacts with DBC1, with consequent abrogation of DBC1 inhibition of SUV39H1, a H3K9 methyltransferase, suggesting the mode of KLLN regulating H3K9me3. These results suggest a critical role for KLLN as a potential regulator of pericentric heterochromatin formation, genomic stability and gene expression. PMID:26673699

  20. Biochemical Analysis of Autophagy in Algae and Plants by Monitoring the Electrophoretic Mobility of ATG8.

    Science.gov (United States)

    Pérez-Pérez, María Esther; Andrés-Garrido, Ascensión; Crespo, José L

    2016-01-01

    Identification of specific autophagy markers has been fundamental to investigate autophagy as catabolic process. Among them, the ATG8 protein turned out to be one of the most widely used and specific molecular markers of autophagy both in higher and lower eukaryotes. Here, we describe how ATG8 can be used to monitor autophagy in Chlamydomonas and Arabidopsis by western blot analysis. PMID:27424752

  1. Autophagy: Friend or Foe in Breast Cancer Development, Progression, and Treatment

    Directory of Open Access Journals (Sweden)

    Damian E. Berardi

    2011-01-01

    Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings.

  2. Adiponectin stimulates autophagy and reduces oxidative stress to enhance insulin sensitivity during high-fat diet feeding in mice.

    Science.gov (United States)

    Liu, Ying; Palanivel, Rengasamy; Rai, Esther; Park, Min; Gabor, Tim V; Scheid, Michael P; Xu, Aimin; Sweeney, Gary

    2015-01-01

    Numerous studies have characterized the antidiabetic effects of adiponectin, yet the precise cellular mechanisms in skeletal muscle, in particular, changes in autophagy, require further clarification. In the current study, we used a high-fat diet (HFD) to induce obesity and insulin resistance in wild-type (WT) or adiponectin knockout (Ad-KO) mice with and without adiponectin replenishment. Temporal analysis of glucose tolerance and insulin sensitivity using hyperinsulinemic-euglycemic clamp and muscle insulin receptor substrate and Akt phosphorylation demonstrated exaggerated and more rapid HFD-induced insulin resistance in skeletal muscle of Ad-KO mice. Superoxide dismutase activity, the reduced glutathione-to-glutathione disulfide ratio, and lipid peroxidation indicated that HFD-induced oxidative stress was corrected by adiponectin. Gene array analysis implicated several antioxidant enzymes, including Gpxs, Prdx, Sod, and Nox4, in mediating this effect. Adiponectin also attenuated palmitate-induced reactive oxygen species production in cultured myotubes and improved insulin-stimulated glucose uptake in primary muscle cells. Increased LC3-II and decreased p62 expression suggested that HFD induced autophagy in muscle of WT mice; however, these changes were not observed in Ad-KO mice. Replenishing adiponectin in Ad-KO mice increased LC3-II and Beclin1 and decreased p62 protein levels, induced fibroblast growth factor-21 expression, and corrected HFD-induced decreases in LC3, Beclin1, and ULK1 gene expression. In vitro studies examining changes in phospho-ULK1 (Ser555), LC3-II, and lysosomal enzyme activity confirmed that adiponectin directly induced autophagic flux in cultured muscle cells in an AMPK-dependent manner. We overexpressed an inactive mutant of Atg5 to create an autophagy-deficient cell model, and together with pharmacological inhibition of autophagy, demonstrated reduced insulin sensitivity under these conditions. In summary, adiponectin stimulated

  3. Opening new doors in autophagy research: Patrice Codogno.

    Science.gov (United States)

    Codogno, Patrice; Klionsky, Daniel J

    2016-06-01

    Patrice Codogno ( Fig. 1 ), one of the associate editors of Autophagy since it was established, is well known in the autophagy field, and has played a particularly important role in France, serving as the first president of Club Francophone de l'AuTophaGie (CFATG). Patrice's research career spans from the predominantly biochemical analyses that were commonly used in the 1980s to the molecular studies that are the primary focus of many labs currently studying autophagy today. Anyone who has met Patrice knows that he is modest, which means his contributions to autophagy and to promoting the careers of scientists globally, are underappreciated. In addition, there is a fun-loving side to Patrice that is often hidden to the casual observer, and it is time to share some of his personality and thoughts with the rest of the autophagy community. PMID:27158743

  4. Role of autophagy in the regulation of epithelial cell junctions.

    Science.gov (United States)

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  5. Autophagy in Plants--What's New on the Menu?

    Science.gov (United States)

    Michaeli, Simon; Galili, Gad; Genschik, Pascal; Fernie, Alisdair R; Avin-Wittenberg, Tamar

    2016-02-01

    Autophagy is a major cellular degradation pathway in eukaryotes. Recent studies have revealed the importance of autophagy in many aspects of plant life, including seedling establishment, plant development, stress resistance, metabolism, and reproduction. This is manifested by the dual ability of autophagy to execute bulk degradation under severe environmental conditions, while simultaneously to be highly selective in targeting specific compartments and protein complexes to regulate key cellular processes, even during favorable growth conditions. Delivery of cellular components to the vacuole enables their recycling, affecting the plant metabolome, especially under stress. Recent research in Arabidopsis has further unveiled fundamental mechanistic aspects in autophagy which may have relevance in non-plant systems. We review the most recent discoveries concerning autophagy in plants, touching upon all these aspects. PMID:26598298

  6. Modulation of Autophagy-Like Processes by Tumor Viruses

    Directory of Open Access Journals (Sweden)

    Karl Munger

    2012-06-01

    Full Text Available Autophagy is an intracellular degradation pathway for long-lived proteins and organelles. This process is activated above basal levels upon cell intrinsic or environmental stress and dysregulation of autophagy has been linked to various human diseases, including those caused by viral infection. Many viruses have evolved strategies to directly interfere with autophagy, presumably to facilitate their replication or to escape immune detection. However, in some cases, modulation of autophagy appears to be a consequence of the virus disturbing the cell’s metabolic signaling networks. Here, we summarize recent advances in research at the interface of autophagy and viral infection, paying special attention to strategies that human tumor viruses have evolved.

  7. Changes of beclin 1 expression in radiation-induced autophagy in breast cancer cells

    International Nuclear Information System (INIS)

    Objective: To investigate the changes of beclin 1 expression in radiation-induced autophagy in breast cancer cell line MCF-7. Methods: Real-time RT-PCR was used to detect the expression of autophagy-related genes MAP1LC3B. Reverse transcription-PCR was used to detect the expression of beclin 1. Western blot was used to analyze the protein expression of beclin 1. Results: Dose-response of MAP1LC3B mRNA studies showed that MAP1LC3B mRNA increased in 8, 16 and 32 h dose-effect studies, up to peak at 12 Gy in 8 h study (F=13.831, P<0.05), 12 Gy in 16 h study (F=10.996, P<0.01) and 8 Gy in 32 h study (F=17.019, P<0.01), respectively. Time-response studies showed that MAP1LC3B mRNA increased and reached to peak at 16 h in 2 Gy dose-effect study (F=16.284, P<0.01), 32 h in 8 Gy study (F=9.030, P<0.05) and 16 h in 12 Gy study (F=20.315, P<0.05), respectively. Dose-response studies of beclin 1 mRNA showed that beclin 1 increased after 2, 4 and 8 Gy irradiation. Time-response studies showed that beclin 1 increased in a time-dependent manner after 8 Gy irradiation. 16 h dose-effect study of beclin 1 protein showed the increase of beclin 1 expression, and 2 Gy time-response study that beclin 1 expression increased and reached the peak at 8 h . Conclusions: X-ray irradiation could induce autophagy in MCF-7 cells. beclin 1 might play an important role in the process of autophagy. (authors)

  8. Skeletal muscle homeostasis in Duchenne muscular dystrophy: modulating autophagy as a promising therapeutic strategy

    Directory of Open Access Journals (Sweden)

    Clara eDe Palma

    2014-07-01

    Full Text Available Muscular dystrophies are a group of genetic and heterogeneous neuromuscular disorders characterised by the primary wasting of skeletal muscle. In Duchenne muscular dystrophy (DMD, the most severe form of these diseases, the mutations in the dystrophin gene lead to muscle weakness and wasting, exhaustion of muscular regenerative capacity and chronic local inflammation leading to substitution of myofibres by connective and adipose tissue. DMD patients suffer of continuous and progressive skeletal muscle damage followed by complete paralysis and death, usually by respiratory and/or cardiac failure. No cure is yet available, but several therapeutic approaches aiming at reversing the ongoing degeneration have been investigated in preclinical and clinical settings. The autophagy is an important proteolytic system of the cell and has a crucial role in the removal of proteins, aggregates and organelles. Autophagy is constantly active in skeletal muscle and its role in tissue homeostasis is complex: at high levels it can be detrimental and contribute to muscle wasting; at low levels it can cause weakness and muscle degeneration, due to the unchecked accumulation of damaged proteins and organelles. The causal relationship between DMD pathogenesis and dysfunctional autophagy has been recently investigated. At molecular levels, the Akt axis is one of the key disregulated pathways, although the molecular events are not completely understood.The aim of this review is to describe and discuss the clinical relevance of the recent advances dissecting autophagy and its signalling pathway in DMD. The picture might pave the way for the development of interventions that are able to boost muscle growth and/or prevent muscle wasting.

  9. Distribution of Salmonella paratyphi A outer membrane protein X gene and immune-protective effect related to its recombinant expressed products

    Institute of Scientific and Technical Information of China (English)

    李明雷

    2014-01-01

    Objective To determine the distribution and sequence conservation of outer membrane protein X(ompX)gene in Salmonella paratyphi A isolates as well as the immunogenicity and immono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity

  10. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

    OpenAIRE

    Nijhof, A. M.; Taoufik, A.; de la Fuente, M.R.; Kocan, K M; de Vries, E; Jongejan, F

    2007-01-01

    The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing w...

  11. GAIP interacting protein C-terminus regulates autophagy and exosome biogenesis of pancreatic cancer through metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Santanu Bhattacharya

    Full Text Available GAIP interacting protein C terminus (GIPC is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance