WorldWideScience

Sample records for autophagy genes protect

  1. The protective roles of autophagy in ischemic preconditioning

    Institute of Scientific and Technical Information of China (English)

    Wen-jun YAN; Hai-long DONG; Li-ze XIONG

    2013-01-01

    Autophagy,a process for the degradation of protein aggregates and dysfunctional organelles,is required for cellular homeostasis and cell survival in response to stress and is implicated in endogenous protection.Ischemic preconditioning is a brief and nonlethal episode of ischemia,confers protection against subsequent ischemia-repenfusion through the up-regulation of endogenous protective mechanisms.Emerging evidence shows that autophagy is associated with the protective effect of ischemic preconditioning.This review summarizes recent progress in research on the functions and regulations of the autophagy pathway in preconditioning-induced protection and cellular survival.

  2. Overexpression of Annexin II Receptor-Induced Autophagy Protects Against Apoptosis in Uveal Melanoma Cells.

    Science.gov (United States)

    Zhang, Yuelu; Song, Hongyuan; Guo, Ting; Zhu, Yongzhe; Tang, Hailin; Qi, Zhongtian; Zhao, Ping; Zhao, Shihong

    2016-05-01

    Uveal melanoma is the most common primary malignant intraocular tumor in adults and still lacks effective systemic therapies. Annexin A2 receptor (AXIIR), a receptor for Annexin II, was demonstrated to play an important role in multiple cells, but its role in uveal melanoma cells remains exclusive. Herein, the authors reported that overexpression of AXIIR was able to reduce cell viability and activate apoptosis apparently in the Mum2C uveal melanoma cell line. Meanwhile, overexpression of AXIIR could induce autophagy and increase autophagy flux. After autophagy was inhibited by chloroquine, enhanced apoptosis and cytotoxicity could be detected. In summary, these data highlighted the crucial role of AXIIR in reducing Mum2C cell viability through inducing apoptosis, while autophagy played a protective role in this process. Interference of this gene may be a promising method for uveal melanoma therapy and combination with specific inhibitor of autophagy may serve as a supplementary.

  3. Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification.

    Science.gov (United States)

    Xu, Hong-guang; Yu, Yun-fei; Zheng, Quan; Zhang, Wei; Wang, Chuang-dong; Zhao, Xiao-yn; Tong, Wen-xue; Wang, Hong; Liu, Ping; Zhang, Xiao-ling

    2014-09-01

    Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification.

  4. Basal autophagy protects cardiomyocytes from doxorubicin-induced toxicity.

    Science.gov (United States)

    Pizarro, Marcela; Troncoso, Rodrigo; Martínez, Gonzalo J; Chiong, Mario; Castro, Pablo F; Lavandero, Sergio

    2016-08-31

    Doxorubicin (Doxo) is one of the most effective anti-neoplastic agents but its cardiotoxicity has been an important clinical limitation. The major mechanism of Doxo-induced cardiotoxicity is associated to its oxidative capacity. However, other processes are also involved with significant consequences for the cardiomyocyte. In recent years, a number of studies have investigated the role of autophagy on Doxo-induced cardiotoxicity but to date it is not clear how Doxo alters that process and its consequence on cardiomyocytes viability. Here we investigated the effect of Doxo 1uM for 24h of stimulation on cultured neonatal rat cardiomyocytes. We showed that Doxo inhibits basal autophagy. This inhibition is due to both Akt/mTOR signaling pathway activation and Beclin 1 level decrease. To assess the role of autophagy on Doxo-induced cardiomyocyte death, we evaluated the effects 3-methyladenine (3-MA), bafilomycin A1 (BafA), siRNA Beclin 1 (siBeclin 1) and rapamycin (Rapa) on cell viability. Inhibition of autophagy with 3-MA, BafA and siBeclin 1 increased lactate dehydrogenase (LDH) release but, when autophagy was induced by Rapa, Doxo-induced cardiomyocyte death was decreased. These results suggest that Doxo inhibits basal autophagy and contributes to cardiomyocyte death. Activation of autophagy could be used as a strategy to protect the heart against Doxo toxicity.

  5. Crosstalk of clock gene expression and autophagy in aging

    Science.gov (United States)

    Kalfalah, Faiza; Janke, Linda; Schiavi, Alfonso; Tigges, Julia; Ix, Alexander; Ventura, Natascia; Boege, Fritz; Reinke, Hans

    2016-01-01

    Autophagy and the circadian clock counteract tissue degeneration and support longevity in many organisms. Accumulating evidence indicates that aging compromises both the circadian clock and autophagy but the mechanisms involved are unknown. Here we show that the expression levels of transcriptional repressor components of the circadian oscillator, most prominently the human Period homologue PER2, are strongly reduced in primary dermal fibroblasts from aged humans, while raising the expression of PER2 in the same cells partially restores diminished autophagy levels. The link between clock gene expression and autophagy is corroborated by the finding that the circadian clock drives cell-autonomous, rhythmic autophagy levels in immortalized murine fibroblasts, and that siRNA-mediated downregulation of PER2 decreases autophagy levels while leaving core clock oscillations intact. Moreover, the Period homologue lin-42 regulates autophagy and life span in the nematode Caenorhabditis elegans, suggesting an evolutionarily conserved role for Period proteins in autophagy control and aging. Taken together, this study identifies circadian clock proteins as set-point regulators of autophagy and puts forward a model, in which age-related changes of clock gene expression promote declining autophagy levels. PMID:27574892

  6. Analysis of autophagy genes in microalgae: Chlorella as a potential model to study mechanism of autophagy.

    Directory of Open Access Journals (Sweden)

    Qiao Jiang

    Full Text Available BACKGROUND: Microalgae, with the ability to mitigate CO(2 emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary. PRINCIPAL FINDINGS: In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen "core autophagy machinery" proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including "receptor-adaptor" complexes in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the "core autophagy machinery" and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor could suppress the formation of autophagic vacuoles in Chlorella. CONCLUSIONS: Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in

  7. Polymorphisms in autophagy genes and susceptibility to tuberculosis.

    NARCIS (Netherlands)

    Songane, M.; Kleinnijenhuis, J.; Alisjahbana, B.; Sahiratmadja, E.; Parwati, I.; Oosting, M.; Plantinga, T.S.; Joosten, L.A.B.; Netea, M.G.; Ottenhoff, T.H.; Vosse, E. van de; Crevel, R. van

    2012-01-01

    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

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

    Science.gov (United States)

    Han, Xiao; Liu, Jian-xun; Li, Xin-zhi

    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 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, 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. Moreover, 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-II 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-II, 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. PMID:21113177

  9. Lifespan extension by suppression of autophagy genes in Caenorhabditis elegans.

    Science.gov (United States)

    Hashimoto, Yasufumi; Ookuma, Sadatsugu; Nishida, Eisuke

    2009-06-01

    Lifespan is regulated by a complex combination of environmental and genetic factors. Autophagy, which is a bulk degradation system of macromolecules and organelles, has an important role in various biological events. In Caenorhabditis elegans, several autophagy genes have been shown to have a role in promoting longevity, but many other autophagy genes have not been examined for their role in the lifespan regulation. Here we have systematically examined the effect of RNAi suppression of 14 autophagy genes on lifespan. While maternal RNAi of autophagy genes in wild-type worms tended to reduce lifespan, maternal RNAi of each of seven autophagy genes in the insulin/IGF-1 receptor daf-2 mutants extended lifespan. Remarkably, RNAi of unc-51/atg-1, bec-1/atg-6 or atg-9, from young adult, i.e. after development, extended lifespan in both wild-type animals and daf-2 mutants, although RNAi of one or two genes shortened it. Moreover, our analysis suggests that the lifespan extension, which is induced by RNAi of unc-51, bec-1 or atg-9 after development, does not require the transcription factor daf-16, the NAD(+)-dependent protein deacetylase sir-2.1 or the genes related to mitochondrial functions. Collectively, our results suggest that autophagy may not always be beneficial to longevity, but may also function to restrict lifespan in C. elegans.

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

    OpenAIRE

    Han, Xiao; Liu, Jian-Xun; Xin-zhi LI

    2010-01-01

    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 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, the abundance of LC3-positive fluoresce...

  11. 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.

  12. Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus.

    Science.gov (United States)

    Tsuboi, Koichiro; Nishitani, Mayo; Takakura, Atsushi; Imai, Yasuyuki; Komatsu, Masaaki; Kawashima, Hiroto

    2015-08-14

    Genome-wide association studies of inflammatory bowel diseases identified susceptible loci containing an autophagy-related gene. However, the role of autophagy in the colon, a major affected area in inflammatory bowel diseases, is not clear. Here, we show that colonic epithelial cell-specific autophagy-related gene 7 (Atg7) conditional knock-out (cKO) mice showed exacerbation of experimental colitis with more abundant bacterial invasion into the colonic epithelium. Quantitative PCR analysis revealed that cKO mice had abnormal microflora with an increase of some genera. Consistently, expression of antimicrobial or antiparasitic peptides such as angiogenin-4, Relmβ, intelectin-1, and intelectin-2 as well as that of their inducer cytokines was significantly reduced in the cKO mice. Furthermore, secretion of colonic mucins that function as a mucosal barrier against bacterial invasion was also significantly diminished in cKO mice. Taken together, our results indicate that autophagy in colonic epithelial cells protects against colitis by the maintenance of normal gut microflora and secretion of mucus.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Vanessa M. Hubbard

    2011-07-01

    Full Text Available 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, autophagy genes, and Crohn’s disease and discuss how host-pathogen interactions can mediate complex inflammatory disorders.

  15. 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.

  16. 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.

  17. Stimulation of autophagy by the p53 target gene Sestrin2.

    Science.gov (United States)

    Maiuri, Maria Chiara; Malik, Shoaib Ahmad; Morselli, Eugenia; Kepp, Oliver; Criollo, Alfredo; Mouchel, Pierre-Luc; Carnuccio, Rosa; Kroemer, Guido

    2009-05-15

    The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

  18. 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 rategenes 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.

  19. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  20. 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/...

  1. 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.

  2. Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro.

    Science.gov (United States)

    Jiang, Xu-Shun; Chen, Xue-Mei; Wan, Jiang-Min; Gui, Hai-Bo; Ruan, Xiong-Zhong; Du, Xiao-Gang

    2017-02-22

    Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis.

  3. 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.

  4. Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro

    Science.gov (United States)

    Jiang, Xu-shun; Chen, Xue-mei; Wan, Jiang-min; Gui, Hai-bo; Ruan, Xiong-zhong; Du, Xiao-gang

    2017-01-01

    Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis. PMID:28225005

  5. Autophagy protects against de novo formation of the [PSI+] prion in yeast.

    Science.gov (United States)

    Speldewinde, Shaun H; Doronina, Victoria A; Grant, Chris M

    2015-12-15

    Prions are self-propagating, infectious proteins that underlie several neurodegenerative diseases. The molecular basis underlying their sporadic formation is poorly understood. We show that autophagy protects against de novo formation of [PSI(+)], which is the prion form of the yeast Sup35 translation termination factor. Autophagy is a cellular degradation system, and preventing autophagy by mutating its core components elevates the frequency of spontaneous [PSI(+)] formation. Conversely, increasing autophagic flux by treating cells with the polyamine spermidine suppresses prion formation in mutants that normally show a high frequency of de novo prion formation. Autophagy also protects against the de novo formation of another prion, namely the Rnq1/[PIN(+)] prion, which is not related in sequence to the Sup35/[PSI(+)] prion. We show that growth under anaerobic conditions in the absence of molecular oxygen abrogates Sup35 protein damage and suppresses the high frequency of [PSI(+)] formation in an autophagy mutant. Autophagy therefore normally functions to remove oxidatively damaged Sup35, which accumulates in cells grown under aerobic conditions, but in the absence of autophagy, damaged/misfolded Sup35 undergoes structural transitions favoring its conversion to the propagatable [PSI(+)] form.

  6. 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.

  7. 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.

  8. Sinomenine hydrochloride protects against polymicrobial sepsis via autophagy.

    Science.gov (United States)

    Jiang, Yu; Gao, Min; Wang, Wenmei; Lang, Yuejiao; Tong, Zhongyi; Wang, Kangkai; Zhang, Huali; Chen, Guangwen; Liu, Meidong; Yao, Yongming; Xiao, Xianzhong

    2015-01-23

    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.

  9. Control of autophagy by oncogenes and tumor suppressor genes.

    Science.gov (United States)

    Maiuri, M C; Tasdemir, E; Criollo, A; Morselli, E; Vicencio, J M; Carnuccio, R; Kroemer, G

    2009-01-01

    Multiple oncogenes (in particular phosphatidylinositol 3-kinase, PI3K; activated Akt1; antiapoptotic proteins from the Bcl-2 family) inhibit autophagy. Similarly, several tumor suppressor proteins (such as BH3-only proteins; death-associated protein kinase-1, DAPK1; the phosphatase that antagonizes PI3K, PTEN; tuberous sclerosic complex 1 and 2, TSC1 and TSC2; as well as LKB1/STK11) induce autophagy, meaning that their loss reduces autophagy. Beclin-1, which is required for autophagy induction acts as a haploinsufficient tumor suppressor protein, and other essential autophagy mediators (such as Atg4c, UVRAG and Bif-1) are bona fide oncosuppressors. One of the central tumor suppressor proteins, p53 exerts an ambiguous function in the regulation of autophagy. Within the nucleus, p53 can act as an autophagy-inducing transcription factor. Within the cytoplasm, p53 exerts a tonic autophagy-inhibitory function, and its degradation is actually required for the induction of autophagy. The role of autophagy in oncogenesis and anticancer therapy is contradictory. Chronic suppression of autophagy may stimulate oncogenesis. However, once a tumor is formed, autophagy inhibition may be a therapeutic goal for radiosensitization and chemosensitization. Altogether, the current state-of-the art suggests a complex relationship between cancer and deregulated autophagy that must be disentangled by further in-depth investigation.

  10. 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.

  11. Remote ischemic preconditioning protects against liver ischemia-reperfusion injury via heme oxygenase-1-induced autophagy.

    Directory of Open Access Journals (Sweden)

    Yun Wang

    Full Text Available BACKGROUND: Growing evidence has linked autophagy to a protective role of preconditioning in liver ischemia/reperfusion (IR. Heme oxygenase-1 (HO-1 is essential in limiting inflammation and preventing the apoptotic response to IR. We previously demonstrated that HO-1 is up-regulated in liver graft after remote ischemic preconditioning (RIPC. The aim of this study was to confirm that RIPC protects against IR via HO-1-mediated autophagy. METHODS: RIPC was performed with regional ischemia of limbs before liver ischemia, and HO-1 activity was inhibited pre-operation. Autophagy was assessed by the expression of light chain 3-II (LC3-II. The HO-1/extracellular signal-related kinase (ERK/p38/mitogen-activated protein kinase (MAPK pathway was detected in an autophagy model and mineral oil-induced IR in vitro. RESULTS: In liver IR, the expression of LC3-II peaked 12-24 h after IR, and the ultrastructure revealed abundant autophagosomes in hepatocytes after IR. Autophagy was inhibited when HO-1 was inactivated, which we believe resulted in the aggravation of liver IR injury (IRI in vivo. Hemin-induced autophagy also protected rat hepatocytes from IRI in vitro, which was abrogated by HO-1 siRNA. Phosphorylation of p38-MAPK and ERK1/2 was up-regulated in hemin-pretreated liver cells and down-regulated after treatment with HO-1 siRNA. CONCLUSIONS: RIPC may protect the liver from IRI by induction of HO-1/p38-MAPK-dependent autophagy.

  12. 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.

  13. Curcumin induces apoptosis and protective autophagy in castration-resistant prostate cancer cells through iron chelation

    Science.gov (United States)

    Yang, Chunguang; Ma, Xueyou; Wang, Zhihua; Zeng, Xing; Hu, Zhiquan; Ye, Zhangqun; Shen, Guanxin

    2017-01-01

    Background Curcumin induces apoptosis and autophagy in different cancer cells. Moreover, chemical and biological experiments have evidenced that curcumin is a biologically active iron chelator and induces cytotoxicity through iron chelation. We thus hypothesized that curcumin may induce apoptosis and autophagy in castration-resistant prostate cancer (CRPC) cells through its iron-chelating properties. Materials and methods CRPC cells were loaded with curcumin alone or in combination with ferric ammonium citrate (FAC). Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by flow cytometry, terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay and caspase activity. Autophagy status was analyzed by the detection of autophagosomes and light chain 3-II (LC3-II) using transmission electron microscopy and Western blot. Iron-binding activity of curcumin was assessed by spectrophotometry and MTT assay. The expression levels of transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) were examined by Western blot. Results Curcumin induced apoptosis and autophagy in CRPC cells. Combining curcumin with autophagy inhibitors (3-methyladenine [3-MA]) synergized the apoptotic effect of curcumin. Moreover, curcumin bound to FAC at a ratio of ~1:1, as assessed by spectrophotometry and MTT assay. Apoptosis and autophagy induced by curcumin were counteracted by equal amounts of FAC. At apoptosis- and autophagy-inducing concentrations, curcumin enhanced the expression levels of TfR1 and IRP1, indicative of iron deprivation induced by curcumin. Conclusion Together, our results indicate that curcumin induces apoptosis and protective autophagy in CRPC cells, which are at least partially dependent on its iron-chelating properties. PMID:28243065

  14. The influence of autophagy on mouse inflammatory responses caused by Salmonella enterica serovar Typhimurium with spv genes

    Institute of Scientific and Technical Information of China (English)

    LI Yuan-Yuan; WU Shu-Yan; CHU Yuan-Yuan; LIAO LI; LIQiong; HUANG Rui

    2011-01-01

    An investigation into the effects of Salmonella plasmid virulence genes (spv) on autophagy,apoptosis,and inflammation was carried out in mice,using a strain of Salmonella enterica serovar Typhimurium (S.typhimurium) SR-11 carrying spv.Strain BRD509 without spy was used as a control.Results showed that the expression of autophagy protein Beclin-1 in the livers and spleens in the SR-11 group was lower than that in the BRD509 group,while the apoptosis protein,Caspase-3,was higher in the SR-11 group.Inflammatory cytokine levels [interleukin 12 (IL-12) and interferon γ (IFN-γ)] were higher in the SR-11 group compared with those in the BRD509 group since 4 d post-infection.In addition,we found an increase in severe pathological changes and larger viable bacterial amounts in livers and spleens in the SR-11 group.After intervention with autophagy agonist rapamycin (RAPA),Beclin-1 expression increased in both groups,while Caspase-3 expression was different between the two groups: Caspase-3 decreased in the SR-11 group but increased in the BRD509 group.Moreover,RAPA decreased cytokine levels,bacterial quantity and organ-related injury in the SR-11 group whereas RAPA increased cytokine levels and aggravated organ injury in the BRD509 group.Results from these studies suggest that S.typhimurium with spv genes may exacerbate infection by inhibiting autophagy and affecting the production of inflammatory cytokines.RAPA-enhanced autophagy may improve the secretion of cytokines in order to protect the host from damaging by Salmonella infection.Our study suggests that the regulation of cellular autophagy may play a role in the prevention and control of certain infectious diseases.

  15. 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.

  16. An autophagy gene, HoATG5, is involved in sporulation, cell wall integrity and infection of wounded barley leaves.

    Science.gov (United States)

    Liu, Ning; Ning, Guo-Ao; Liu, Xiao-Hong; Feng, Xiao-Xiao; Lu, Jian-Ping; Mao, Li-Juan; Su, Zhen-Zhu; Wang, Ying; Zhang, Chu-Long; Lin, Fu-Cheng

    2016-11-01

    The endophytic fungus Harpophora oryzae is a beneficial endosymbiont isolated from wild rice. H. oryzae can not only promote rice growth and biomass accumulation but also protect rice roots from invasion by its close relative Magnaporthe oryzae. Autophagy is a highly evolutionary conserved process from lower to higher eukaryotic organisms, and is involved in the maintenance of normal cell differentiation and development. In this study, we isolated a gene (HoATG5) which encodes an essential protein required for autophagy from the beneficial endophyte fungus H. oryzae. Using targeted gene replacement, a ΔHoATG5 mutant was generated and used to investigate the biological functions of autophagy in H. oryzae. We found that the autophagic process was blocked in the HoATG5 deletion mutant. The mutant showed increased vegetative growth and sporulation, and was sensitive to nutrient starvation. The ΔHoATG5 mutant lost its ability to penetrate and infect the wounded barley leaves. These results provide new knowledge to elaborate the molecular machinery of autophagy in endophytic fungi.

  17. Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis.

    Science.gov (United States)

    Wang, Zhen-Yu; Lin, Jian-Hua; Muharram, Akram; Liu, Wen-Ge

    2014-06-01

    Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1 expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increased Bcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.

  18. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis

    Science.gov (United States)

    Karantza-Wadsworth, Vassiliki; Patel, Shyam; Kravchuk, Olga; Chen, Guanghua; Mathew, Robin; Jin, Shengkan; White, Eileen

    2007-01-01

    Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression. PMID:17606641

  19. Autophagy protects cardiomyocytes from the myocardial ischaemia-reperfusion injury through the clearance of CLP36

    Science.gov (United States)

    Li, Shiguo; Liu, Chao; Gu, Lei; Wang, Lina; Shang, Yongliang; Liu, Qiong; Wan, Junyi; Shi, Jian; Wang, Fang; Xu, Zhiliang; Ji, Guangju

    2016-01-01

    Cardiovascular disease (CVD) is the leading cause of the death worldwide. An increasing number of studies have found that autophagy is involved in the progression or prevention of CVD. However, the precise mechanism of autophagy in CVD, especially the myocardial ischaemia-reperfusion injury (MI/R injury), is unclear and controversial. Here, we show that the cardiomyocyte-specific disruption of autophagy by conditional knockout of Atg7 leads to severe contractile dysfunction, myofibrillar disarray and vacuolar cardiomyocytes. A negative cytoskeleton organization regulator, CLP36, was found to be accumulated in Atg7-deficient cardiomyocytes. The cardiomyocyte-specific knockout of Atg7 aggravates the MI/R injury with cardiac hypertrophy, contractile dysfunction, myofibrillar disarray and severe cardiac fibrosis, most probably due to CLP36 accumulation in cardiomyocytes. Altogether, this work reveals autophagy may protect cardiomyocytes from the MI/R injury through the clearance of CLP36, and these findings define a novel relationship between autophagy and the regulation of stress fibre in heart. PMID:27512143

  20. Autophagy induced by p53-reactivating molecules protects pancreatic cancer cells from apoptosis.

    Science.gov (United States)

    Fiorini, Claudia; Menegazzi, Marta; Padroni, Chiara; Dando, Ilaria; Dalla Pozza, Elisa; Gregorelli, Alex; Costanzo, Chiara; Palmieri, Marta; Donadelli, Massimo

    2013-03-01

    TP53 mutations compromising p53 transcriptional function occur in more than 50 % of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1-2/AMPK/mTOR by inducing AMPK phosphorylation on Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize both pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53-reactivating molecules, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.

  1. Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR-dependent pathway.

    Science.gov (United States)

    Jiang, LiBo; Jin, YongLong; Wang, HuiRen; Jiang, YunQi; Dong, Jian

    2014-11-01

    Although glucosamine has been suggested to be effective in the treatment of osteoarthritis, its effect on disc degeneration remains unclear. We sought to explore whether glucosamine can activate autophagy in rat nucleus pulposus (NP) cells and protect cells treated with IL-1β or hydrogen peroxide (H2 O2 ). Autophagy in cells was examined by detecting for LC3, Beclin-1, m-TOR, and p70S6K, as well as by analyzing autophagosomes. To inhibit autophagy, 3-methyladenine (3-MA) was used. In the cells treated with IL-1β, the levels of Adamts-4, Mmp-13, aggrecan, and Col2a1 were analyzed by real-time PCR and immunofluorescence. Apoptosis was analyzed by TUNEL. Cell senescence under H2 O2 was revealed by SA-β-Gal staining. Glucosamine could activate autophagy in a dose-dependent manner within 24 h and inhibit the phosphorylation of m-TOR and p70S6K. Autophagy in IL-1β or H2 O2 -treated cells was increased by glucosamine. Glucosamine attenuated the decrease of aggrecan and prevented the apoptosis of the NP cells induced by IL-1β, whereas 3-MA partly reversed these effects. The percentage of SA-β-Gal-positive cells induced by H2 O2 treatment was decreased by glucosamine, accompanied by the decline of p70S6K phosphorylation. Glucosamine protects NP cells and up-regulates autophagy by inhibiting the m-TOR pathway, which might point a potential therapeutic agent for disc degeneration.

  2. Transcriptional activation of TFEB/ZKSCAN3 target genes underlies enhanced autophagy in spinobulbar muscular atrophy.

    Science.gov (United States)

    Chua, Jason P; Reddy, Satya L; Merry, Diane E; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Robins, Diane M; Lieberman, Andrew P

    2014-03-01

    Spinobulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in exon 1 of the androgen receptor (AR) gene. SBMA demonstrates androgen-dependent toxicity due to unfolding and aggregation of the mutant protein. There are currently no disease-modifying therapies, but of increasing interest for therapeutic targeting is autophagy, a highly conserved cellular process mediating protein quality control. We have previously shown that genetic manipulations inhibiting autophagy diminish skeletal muscle atrophy and extend the lifespan of AR113Q knock-in mice. In contrast, manipulations inducing autophagy worsen muscle atrophy, suggesting that chronic, aberrant upregulation of autophagy contributes to pathogenesis. Since the degree to which autophagy is altered in SBMA and the mechanisms responsible for such alterations are incompletely defined, we sought to delineate autophagic status in SBMA using both cellular and mouse models. Here, we confirm that autophagy is induced in cellular and knock-in mouse models of SBMA and show that the transcription factors transcription factor EB (TFEB) and ZKSCAN3 operate in opposing roles to underlie these changes. We demonstrate upregulation of TFEB target genes in skeletal muscle from AR113Q male mice and SBMA patients. Furthermore, we observe a greater response in AR113Q mice to physiological stimulation of autophagy by both nutrient starvation and exercise. Taken together, our results indicate that transcriptional signaling contributes to autophagic dysregulation and provides a mechanistic framework for the pathologic increase of autophagic responsiveness in SBMA.

  3. Autophagy and ethanol neurotoxicity.

    Science.gov (United States)

    Luo, Jia

    2014-01-01

    Excessive ethanol exposure is detrimental to the brain. The developing brain is particularly vulnerable to ethanol such that prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD). Neuronal loss in the brain is the most devastating consequence and is associated with mental retardation and other behavioral deficits observed in FASD. Since alcohol consumption during pregnancy has not declined, it is imperative to elucidate the underlying mechanisms and develop effective therapeutic strategies. One cellular mechanism that acts as a protective response for the central nervous system (CNS) is autophagy. Autophagy regulates lysosomal turnover of organelles and proteins within cells, and is involved in cell differentiation, survival, metabolism, and immunity. We have recently shown that ethanol activates autophagy in the developing brain. The autophagic preconditioning alleviates ethanol-induced neuron apoptosis, whereas inhibition of autophagy potentiates ethanol-stimulated reactive oxygen species (ROS) and exacerbates ethanol-induced neuroapoptosis. The expression of genes encoding proteins required for autophagy in the CNS is developmentally regulated; their levels are much lower during an ethanol-sensitive period than during an ethanol-resistant period. Ethanol may stimulate autophagy through multiple mechanisms; these include induction of oxidative stress and endoplasmic reticulum stress, modulation of MTOR and AMPK signaling, alterations in BCL2 family proteins, and disruption of intracellular calcium (Ca2+) homeostasis. This review discusses the most recent evidence regarding the involvement of autophagy in ethanol-mediated neurotoxicity as well as the potential therapeutic approach of targeting autophagic pathways.

  4. 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.

  5. Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    Cao Yang

    2009-05-01

    Full Text Available Abstract Background Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated. Results Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE. In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae. Conclusion Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.

  6. 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.

  7. 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

  8. 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.

  9. Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells.

    Science.gov (United States)

    Zhong, Wu; Zhu, Haichuan; Sheng, Fugeng; Tian, Yonglu; Zhou, Jun; Chen, Yingyu; Li, Song; Lin, Jian

    2014-07-01

    Transition metal copper (Cu) can exist in oxidized or reduced states in cells, leading to cytotoxicity in cancer cells through oxidative stress. Recently, copper complexes are emerging as a new class of anticancer compounds. Here, we report that a novel anticancer copper complex (HYF127c/Cu) induces oxidative stress-dependent cell death in cancer cells. Further, transcriptional analysis revealed that oxidative stress elicits broad transcriptional changes of genes, in which autophagy-related genes are significantly changed in HYF127c/Cu-treated cells. Consistently, autophagy was induced in HYF127c/Cu-treated cells and inhibitors of autophagy promoted cell death induced by HYF127c/Cu. Further analysis identified that the MAPK11/12/13/14 (formerly known as p38 MAPK) pathway was also activated in HYF127c/Cu-treated cells. Meanwhile, the MAPK11/12/13/14 inhibitor SB203580 downregulated autophagy by inhibiting the transcription of the autophagy genes MAP1LC3B, BAG3, and HSPA1A, and promoted HYF127c/Cu-induced cell death. These data suggest that copper-induced oxidative stress will induce protective autophagy through transcriptional regulation of autophagy genes by activation of the MAPK11/12/13/14 pathway in HeLa cells.

  10. Novel functional roles of caspase-related genes in the regulation of apoptosis and autophagy

    Science.gov (United States)

    Shin, Ju-Hyun

    2016-01-01

    Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery. PMID:27847434

  11. Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation.

    Science.gov (United States)

    Datan, E; Roy, S G; Germain, G; Zali, N; McLean, J E; Golshan, G; Harbajan, S; Lockshin, R A; Zakeri, Z

    2016-03-03

    A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress.

  12. 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.

  13. Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin-induced apoptosis by inhibiting cyto-protective autophagy.

    Science.gov (United States)

    He, Jun; Yu, Jing-Jie; Xu, Qing; Wang, Lin; Zheng, Jenny Z; Liu, Ling-Zhi; Jiang, Bing-Hua

    2015-01-01

    Cisplatin is commonly used in ovarian cancer treatment by inducing apoptosis in cancer cells as a result of lethal DNA damage. However, the intrinsic and acquired resistance to cisplatin in cancer cells remains a big challenge for improving overall survival. The cyto-protective functions of autophagy in cancer cells have been suggested as a potential mechanism for chemoresistance. Here, we reported MIR152 as a new autophagy-regulating miRNA that plays a role in cisplatin-resistance. We showed that MIR152 expression was dramatically downregulated in the cisplatin-resistant cell lines A2780/CP70, SKOV3/DDP compared with their respective parental cells, and in ovarian cancer tissues associated with cisplatin-resistance. Overexpression of MIR152 sensitized cisplatin-resistant ovarian cancer cells by reducing cisplatin-induced autophagy, enhancing cisplatin-induced apoptosis and inhibition of cell proliferation. A mouse subcutaneous xenograft tumor model using A2780/CP70 cells with overexpressing MIR152 was established and displayed decreased tumor growth in response to cisplatin. We also identified that ATG14 is a functional target of MIR152 in regulating autophagy inhibition. Furthermore, we found that EGR1 (early growth response 1) regulated the MIR152 gene at the transcriptional level. Ectopic expression of EGR1 enhanced efficacy of chemotherapy in A2780/CP70 cells. More importantly, these findings were relevant to clinical cases. Both EGR1 and MIR152 expression levels were significantly lower in ovarian cancer tissues with high levels of ERCC1 (excision repair cross-complementation group 1), a marker for cisplatin-resistance. Collectively, these data provide insights into novel mechanisms for acquired cisplatin-resistance. Activation of EGR1 and MIR152 may be a useful therapeutic strategy to overcome cisplatin-resistance by preventing cyto-protective autophagy in ovarian cancer.

  14. Sphingosylphosphorylcholine protects cardiomyocytes against ischemic apoptosis via lipid raft/PTEN/Akt1/mTOR mediated autophagy.

    Science.gov (United States)

    Yue, Hong-Wei; Liu, Jing; Liu, Ping-Ping; Li, Wen-Jing; Chang, Fen; Miao, Jun-Ying; Zhao, Jing

    2015-09-01

    Autophagy, evoked by diverse stresses including myocardial ischemia/reperfusion (I/R), profoundly affects the development of heart failure. However, the specific molecular basis of autophagy remains to be elucidated. Here we report that sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, significantly suppressed apoptosis and induced autophagy in cardiomyocytes. Blocking this SPC evoked autophagy by 3-methyladenine (3MA)-sensitized cardiomyocytes to serum deprivation-induced apoptosis. Subsequent studies revealed that SPC downregulated the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) but enhanced that of JNK when inducing autophagy. We identified SPC as a switch for the activity of Akt1, a supposed upstream modulator of both mTOR and JNK. Furthermore, β-cyclodextrin, which destroys membrane cholesterol, abolished the SPC-reduced phosphorylation of both Akt and PTEN, thus inhibiting SPC-induced autophagy. In conclusion, SPC is a novel molecule protecting cardiomyocytes against apoptosis by promoting autophagy. The lipid raft/PTEN/Akt1/mTOR signal pathway is the underlying mechanism and might provide novel targets for cardiac failure therapy.

  15. Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy

    Science.gov (United States)

    Gallo, S; Gatti, S; Sala, V; Albano, R; Costelli, P; Casanova, E; Comoglio, P M; Crepaldi, T

    2014-01-01

    Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl2), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) – surprisingly – autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl2. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury. PMID:24743740

  16. Remifentanil protects human keratinocytes against hypoxia-reoxygenation injury through activation of autophagy.

    Directory of Open Access Journals (Sweden)

    Jae-Young Kwon

    Full Text Available The proliferation, differentiation, and migration of keratinocytes are essential in the early stages of wound healing. Hypoxia-Reoxygenation (H/R injury to keratinocytes can occur in various stressful environments such as surgery, trauma, and various forms of ulcers. The effects of remifentanil on human keratinocytes under hypoxia-reoxygenation have not been fully studied. Therefore, we investigated the effects of remifentanil on the proliferation, apoptosis, and autophagic activation of human keratinocytes during hypoxic-reoxygenation. Human keratinocytes were cultured under 1% oxygen tension for 24 h. The cells were then treated with various concentrations of remifentanil (0.01, 0.1, 0.5, and 1 ng/mL for 2 h. Thereafter, the cells were reoxygenated for 12 h at 37°C. We measured cell viability via MTT assay. Using quantitative real-time PCR and western blot analysis, we measured the expression levels of proteins associated with apoptosis and autophagy. Quantification of apoptotic cells was performed using flow cytometer analysis and autophagic vacuoles were observed under a fluorescence microscope. Remifentanil treatment brought about an increase in the proliferation of human keratinocytes damaged by hypoxia-reoxygenation and decreased the apoptotic cell death, enhancing autophagic activity. However, the autophagy pathway inhibitor 3-MA inhibited the protective effect of remifentanil in hypoxia-reoxygenation injury. In conclusion, the current study demonstrated that remifentanil treatment stimulated autophagy and reduced apoptotic cell death in a hypoxia-reoxygenation model of human keratinocytes. Our results provide additional insights into the relationship between apoptosis and autophagy.

  17. Perifosine induces protective autophagy and upregulation of ATG5 in human chronic myelogenous leukemia cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yin TONG; Yan-yan LIU; Liang-shun YOU; Wen-bin QIAN

    2012-01-01

    Aim:The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML)cells and its mechanisms of action are unknown.In this study,the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML)cell lines were compared to elucidate the mechanisms underlying the differences.Methods:Human AML cell lines Kasumi-1 and HL-60,and the CML cell line K562 were used.Cell viability was quantitated using MTT assay.Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining,which were followed by flow cytometry and fluorescence microscopy analysis,respectively.Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot.Autophagy was studied using electron microscopy,the acridine orange staining method,and GFP-LC3 was examined with fluorescence microscopy.Results:In contrast to AML cell lines,the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line)were resistant to perifosine (2.5-20 μmol/L)in respect to inhibiting cell growth and inducing apoptosis.Perifosine (2.5,5,and 10 μmol/L)inhibited Akt and its phosphorylation in AML cells,but not in CML cells.Treatment with perifosine (20 μmol/L)resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II.Treatment of CML cells with perifosine (5,10,and 20 μmol/L)dose-dependently upregulated AGT5,but not Beclin 1 at the protein level.Furthermore,inhibition of autophagyby chloroquine (40 nmol/L)significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 μmol/L).Conclusion:Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro,which is due to perifosine-induced protective autophagy and upregulation of ATG5.

  18. Hinokitiol protects primary neuron cells against prion peptide-induced toxicity via autophagy flux regulated by hypoxia inducing factor-1.

    Science.gov (United States)

    Moon, Ji-Hong; Lee, Ju-Hee; Lee, You-Jin; Park, Sang-Youel

    2016-05-24

    Prion diseases are fatal neurodegenerative disorders that are derived from structural changes of the native PrPc. Recent studies indicated that hinokitiol induced autophagy known to major function that keeps cells alive under stressful conditions. We investigated whether hinokitiol induces autophagy and attenuates PrP (106-126)-induced neurotoxicity. We observed increase of LC3-II protein level, GFP-LC3 puncta by hinokitiol in neuronal cells. Addition to, electron microscopy showed that hinokitiol enhanced autophagic vacuoles in neuronal cells. We demonstrated that hinokitiol protects against PrP (106-126)-induced neurotoxicity via autophagy by using autophagy inhibitor, wortmannin and 3MA, and ATG5 small interfering RNA (siRNA). We checked hinokitiol activated the hypoxia-inducible factor-1α (HIF-1α) and identified that hinokitiol-induced HIF-1α regulated autophagy. Taken together, this study is the first report demonstrating that hinokitiol protected against prion protein-induced neurotoxicity via autophagy regulated by HIF-1α. We suggest that hinokitiol is a possible therapeutic strategy in neuronal disorders including prion disease.

  19. Cannabidiol protects liver from binge alcohol-induced steatosis by mechanisms including inhibition of oxidative stress and increase in autophagy.

    Science.gov (United States)

    Yang, Lili; Rozenfeld, Raphael; Wu, Defeng; Devi, Lakshmi A; Zhang, Zhenfeng; Cederbaum, Arthur

    2014-03-01

    Acute alcohol drinking induces steatosis, and effective prevention of steatosis can protect liver from progressive damage caused by alcohol. Increased oxidative stress has been reported as one mechanism underlying alcohol-induced steatosis. We evaluated whether cannabidiol, which has been reported to function as an antioxidant, can protect the liver from alcohol-generated oxidative stress-induced steatosis. Cannabidiol can prevent acute alcohol-induced liver steatosis in mice, possibly by preventing the increase in oxidative stress and the activation of the JNK MAPK pathway. Cannabidiol per se can increase autophagy both in CYP2E1-expressing HepG2 cells and in mouse liver. Importantly, cannabidiol can prevent the decrease in autophagy induced by alcohol. In conclusion, these results show that cannabidiol protects mouse liver from acute alcohol-induced steatosis through multiple mechanisms including attenuation of alcohol-mediated oxidative stress, prevention of JNK MAPK activation, and increasing autophagy.

  20. 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.

  1. 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.

  2. 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...

  3. Metformin Protects Against Cisplatin-Induced Tubular Cell Apoptosis and Acute Kidney Injury via AMPKα-regulated Autophagy Induction.

    Science.gov (United States)

    Li, Jianzhong; Gui, Yuan; Ren, Jiafa; Liu, Xin; Feng, Ye; Zeng, Zhifeng; He, Weichun; Yang, Junwei; Dai, Chunsun

    2016-04-07

    Metformin, one of the most common prescriptions for patients with type 2 diabetes, is reported to protect the kidney from gentamicin-induced nephrotoxicity. However, the role and mechanisms for metformin in preventing cisplatin-induced nephrotoxicity remains largely unknown. In this study, a single intraperitoneal injection of cisplatin was employed to induce acute kidney injury (AKI) in CD1 mice. The mice exhibited severe kidney dysfunction and histological damage at day 2 after cisplatin injection. Pretreatment of metformin could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis and inflammatory cell accumulation in the kidneys. Additionally, pretreatment of metformin could enhance both AMPKα phosphorylation and autophagy induction in the kidneys after cisplatin injection. In cultured NRK-52E cells, a rat kidney tubular cell line, metformin could stimulate AMPKα phosphorylation, induce autophagy and inhibit cisplatin-induced cell apoptosis. Blockade of either AMPKα activation or autophagy induction could largely abolish the protective effect of metformin in cisplatin-induced cell death. Together, this study demonstrated that metformin may protect against cisplatin-induced tubular cell apoptosis and AKI through stimulating AMPKα activation and autophagy induction in the tubular cells.

  4. Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population.

    Science.gov (United States)

    Cai, Ping-Ping; Wang, Hong-Xia; Zhuang, Jing-Cong; Liu, Qi-Bing; Zhao, Gui-Xian; Li, Zhen-Xin; Wu, Zhi-Ying

    2014-12-01

    Neuromyelitis optica (NMO) and multiple sclerosis (MS) are autoimmune demyelinating diseases of the central nervous system. The discovery of NMO immunoglobulin G (NMO-IgG) antibody has improved the clinical definition of NMO. Recently, the autophagy-related genes (ATGs) have been proved to be associated with several autoimmune and inflammation diseases. Increased T cell expression of ATG5 may be correlated with the pathogenesis of inflammatory demyelination in MS. However, the association of ATG5 variants with MS and NMO patients has not been well studied. In this study, five ATG5 variants were genotyped in 144 MS patients, 109 NMO patients and 288 controls in the Han Chinese population. In the cohort of NMO patients, we observed that the CC genotype of rs548234 increased susceptibility to NMO (p = 0.016), while the allele T of rs548234 (p = 0.003) and the allele A of rs6937876 (p = 0.009) acted as protective factors for NMO-IgG positive NMO patients. However, no association was found between ATG5 variants and MS patients. These results indicated that ATG5 variants are associated with NMO but not MS patients, which may provide a clue for further clarifying the autoimmune mechanisms of autophagy-related pathogenesis in NMO.

  5. AMP-activated protein kinase-dependent autophagy mediated the protective effect of sonic hedgehog pathway on oxygen glucose deprivation-induced injury of cardiomyocytes.

    Science.gov (United States)

    Xiao, Qing; Yang, Ya; Qin, Yuan; He, Yan-Hua; Chen, Kui-Xiang; Zhu, Jian-Wei; Zhang, Gui-Ping; Luo, Jian-Dong

    2015-02-13

    Sonic hedgehog (Shh) pathway has been reported to protect cardiomyocytes in myocardial infarction (MI), but the underlying mechanism is not clear. Here, we provide evidence that Shh pathway induces cardiomyocytes survival through AMP-activated protein kinase-dependent autophagy. Shh pathway agonist SAG increased the expression of LC3-II, and induced the formation of autophagosomes in cultured H9c2 cardiomyocytes under oxygen glucose deprivation (OGD) 1 h and 4 h. Moreover, SAG induced a profound AMP-activated protein kinase (AMPK) activation, and then directly phosphorylated and activated the downstream autophagy initiator Ulk1, independent of the autophagy suppressor mammalian target of rapamycin (mTOR) complex 1. Taken together, our results have shown that Shh activates AMPK-dependent autophagy in cardiomyocytes under OGD, suggesting a role of autophagy in Shh-induced cellular protection.

  6. Autophagy gene polymorphism is associated with susceptibility to leprosy by affecting inflammatory cytokines.

    Science.gov (United States)

    Yang, Degang; Chen, Jia; Shi, Chao; Jing, Zhichun; Song, Ningjing

    2014-04-01

    Autophagy and inflammation closely interact with each other, and together, they play critical roles in bacterial infection. Leprosy is caused by the infection of Mycobacterium leprae (M. leprae). The objective of the study was to investigate the association between polymorphisms in IRGM, an autophagy gene, and susceptibility to leprosy, and identify possible functions of the polymorphism in the infection of M. leprae. Two polymorphisms in IRGM, rs4958842 and rs13361189, were tested in 412 leprosy cases and 432 healthy controls. Levels of inflammatory cytokines including interleukin 1 beta, IL-4, IL-6, and interferon gamma (INF-γ) were measured after the infection of M. leprae in the peripheral blood mononuclear cell (PBMC) of subjects with different genotypes of rs13361189. Data showed that prevalence of rs13361189TC and CC genotypes were significantly higher in leprosy patients than in healthy controls (odds ratio (OR) = 1.49, 95 % confidence interval (CI) 1.09-2.04, P = 0.012; OR = 2.58, 95 % CI 1.65-4.05, P autophagy gene polymorphism was associated with the increased risk of leprosy by affecting inflammatory cytokines.

  7. Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

    Science.gov (United States)

    Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

    2017-01-01

    Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

  8. Axonal protection by short-term hyperglycemia with involvement of autophagy in TNF-induced optic nerve degeneration

    Directory of Open Access Journals (Sweden)

    Kana eSase

    2015-10-01

    Full Text Available Previous reports showed that short-term hyperglycemia protects optic nerve axons in a rat experimental hypertensive glaucoma model. In this study, we investigated whether short-term hyperglycemia prevents tumor necrosis factor (TNF-induced optic nerve degeneration in rats and examined the role of autophagy in this axon change process. In phosphate-buffered saline-treated rat eyes, no significant difference in axon number between the normoglycemic (NG and streptozotocin-induced hyperglycemic (HG groups was seen at 2weeks. Substantial degenerative changes in the axons were noted 2 weeks after intravitreal injection of TNF in the NG group. However, the HG group showed significant protective effects on axons against TNF-induced optic nerve degeneration compared with the NG group. This protective effect was significantly inhibited by 3-methyladenine, an autophagy inhibitor. Immunoblot analysis showed that the LC3-II level in the optic nerve was increased in the HG group compared with the NG group. Increased p62 protein levels in the optic nerve after TNF injection was observed in the NG group, and this increase was inhibited in the HG group. Electron microscopy showed that autophagosomes were increased in optic nerve axons in the HG group. Immunohistochemical study showed that LC3 was colocalized with nerve fibers in the retina and optic nerve in both the NG and HG groups. Short-term hyperglycemia protects axons against TNF-induced optic nerve degeneration. This axonal-protective effect may be associated with autophagy machinery.

  9. Ampelopsin protects endothelial cells from hyperglycemia-induced oxidative damage by inducing autophagy via the AMPK signaling pathway.

    Science.gov (United States)

    Liang, Xinyu; Zhang, Ting; Shi, Linying; Kang, Chao; Wan, Jing; Zhou, Yong; Zhu, Jundong; Mi, Mantian

    2015-01-01

    Diabetic angiopathy is a major diabetes-specific complication that often begins with endothelial dysfunction induced by hyperglycemia; however, the pathological mechanisms of this progression remain unclear. Ampelopsin is a natural flavonol that has strong antioxidant activity, but little information is available regarding its antidiabetic effect. This study focused on the effect of ampelopsin on hyperglycemia-induced oxidative damage and the underlying mechanism of this effect in human umbilical vein endothelial cells (HUVECs). We found that hyperglycemia impaired autophagy in HUVECs through the inhibition of AMP-activated protein kinase (AMPK), which directly led to endothelial cell damage. Ampelopsin significantly attenuated the detrimental effect of hyperglycemia-induced cell dysfunction in a concentration-dependent manner in HUVECs. Ampelopsin significantly upregulated LC3-II, Beclin1, and Atg5 protein levels but downregulated p62 protein levels in HUVECs. Transmission electron microscopy and confocal microscopy indicated that ampelopsin notably induced autophagosomes and LC3-II dots, respectively. Additionally, the autophagy-specific inhibitor 3-MA, as well as Atg5 and Beclin1 siRNA pretreatment, markedly attenuated ampelopsin-induced autophagy, which subsequently abolished the protective effect of ampelopsin against hyperglycemia in HUVECs. Moreover, ampelopsin also increased AMPK activity and inhibited mTOR (mammalian target of rapamycin) complex activation. Ampelopsin-induced autophagy was attenuated by the AMPK antagonist compound C but strengthened by the AMPK agonist AICAR (5-minoimidazole-4-carboxamide ribonucleotide). Furthermore, AMPK siRNA transfection eliminated ampelopsin's alleviation of cell injury induced by hyperglycemia. The protective effect of ampelopsin against hyperglycemia-induced cell damage, which functions by targeting autophagy via AMPK activation, makes it a promising pharmacological treatment for type-2 diabetes.

  10. Functional variant in the autophagy-related 5 gene promotor is associated with childhood asthma.

    Directory of Open Access Journals (Sweden)

    Lisa J Martin

    Full Text Available RATIONALE AND OBJECTIVE: Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. METHODS: Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. MEASUREMENTS AND MAIN RESULTS: We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively. In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05. We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. CONCLUSION: Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.

  11. Deep hypothermia-enhanced autophagy protects PC12 cells against oxygen glucose deprivation via a mitochondrial pathway.

    Science.gov (United States)

    Tang, Dang; Wang, Cheng; Gao, Yongjun; Pu, Jun; Long, Jiang; Xu, Wei

    2016-10-06

    Deep hypothermia is known for its organ-preservation properties, which is introduced into surgical operations on the brain and heart, providing both safety in stopping circulation as well as an attractive bloodless operative field. However, the molecular mechanisms have not been clearly identified. This study was undertaken to determine the influence of deep hypothermia on neural apoptosis and the potential mechanism of these effects in PC12 cells following oxygen-glucose deprivation. Deep hypothermia (18°C) was given to PC12 cells while the model of oxygen-glucose deprivation (OGD) induction for 1h. After 24h of reperfusion, the results showed that deep hypothermia decreased the neural apoptosis, and significantly suppressed overexpression of Bax, CytC, Caspase 3, Caspase 9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. While deep hypothermia increased the LC3II/LC3I and Beclin 1, an autophagy marker, which can be inhibited by 3-methyladenine (3-MA), indicating that deep hypothermia-enhanced autophagy ameliorated apoptotic cell death in PC12 cells subjected to OGD. Based on these findings we propose that deep hypothermia protects against neural apoptosis after the induction of OGD by attenuating the mitochondrial apoptosis pathway, moreover, the mechanism of these antiapoptosis effects is related to the enhancement of autophagy, which autophagy might provide a means of neuroprotection against OGD.

  12. Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.

    Science.gov (United States)

    Ha, Ji-Young; Kim, Ji-Soo; Kim, Seo-Eun; Son, Jin H

    2014-02-21

    Abnormal autophagy is frequently observed during dopaminergic neurodegeneration in Parkinson's disease (PD). However, it is not yet firmly established whether active autophagy is beneficial or pathogenic with respect to dopaminergic cell loss. Staurosporine, a common inducer of apoptosis, is often used in mechanistic studies of dopaminergic cell death. Here we report that staurosporine activates both autophagy and mitophagy simultaneously during dopaminergic neuronal cell death, and evaluate the physiological significance of these processes during cell death. First, staurosporine treatment resulted in induction of autophagy in more than 75% of apoptotic cells. Pharmacological inhibition of autophagy by bafilomycin A1 decreased significantly cell viability. In addition, staurosporine treatment resulted in activation of the PINK1-Parkin mitophagy pathway, of which deficit underlies some familial cases of PD, in the dopaminergic neuronal cell line, SN4741. The genetic blockade of this pathway by PINK1 null mutation also dramatically increased staurosporine-induced cell death. Taken together, our data suggest that staurosporine induces both mitophagy and autophagy, and that these pathways exert a significant neuroprotective effect, rather than a contribution to autophagic cell death. This model system may therefore be useful for elucidating the mechanisms underlying crosstalk between autophagy, mitophagy, and cell death in dopaminergic neurons.

  13. Potential autophagy enhancers protect against fipronil-induced apoptosis in SH-SY5Y cells.

    Science.gov (United States)

    Park, Jae Hyeon; Lee, Jeong Eun; Lee, Soo-Jin; Park, Soo Jin; Park, Kyung Hun; Jeong, Mihye; Koh, Hyun Chul

    2013-10-23

    Oxidative stress created by environmental toxicants activates several signaling pathways. Autophagy is one of the first lines of defense against oxidative stress damage. The autophagy pathway can be induced and up-regulated in response to intracellular reactive oxygen species (ROS). Recently, we reported that fipronil (FPN)-induced mitochondria-dependent apoptosis is mediated through ROS in human neuroblastoma SH-SY5Y cells. In this study, we explored the role of autophagy to prevent FPN neurotoxicity. We investigated the modulation of FPN-induced apoptosis according to autophagy regulation. FPN activated caspase-9 and caspase-3, and induced nuclear fragmentation and condensation, all of which indicate that FPN-induced cell death was due to apoptosis. In addition, we observed FPN-induced autophagic cell death by monitoring the expression of LC3-II and Beclin-1. Exposure to FPN in SH-SY5Y cells led to the production of ROS. Treatment with N-acetyl-cysteine (NAC) effectively blocked both apoptosis and autophagy. Interestingly, pretreatment with rapamycin, an autophagy inducer, significantly enhanced the viability of FPN-exposed cells; the enhancement of cell viability was partially due to alleviation of FPN-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment with 3-methyladenine (3MA) a specific inhibitor for autophagy, remarkably strengthened FPN toxicity and further induced activation of caspase-3 in these cells. Our studies suggest that FPN-induced cytotoxicity is modified by autophagy regulation and that rapamycin is neuroprotective against FPN-induced apoptosis through enhancing autophagy.

  14. Atorvastatin Protects Vascular Smooth Muscle Cells From TGF-β1-Stimulated Calcification by Inducing Autophagy via Suppression of the β-Catenin Pathway

    Directory of Open Access Journals (Sweden)

    Demin Liu

    2014-01-01

    Full Text Available Background: Arterial calcification is a major event in the progression of atherosclerosis. It is reported that statins exhibit various protective effects against vascular smooth muscle cell (VSMC inflammation and proliferation in cardiovascular remodeling. Although statins counteract atherosclerosis, the molecular mechanisms of statins on the calcium release from VSMCs have not been clearly elucidated. Methods: Calcium content of VSMCs was measured using enzyme-linked immunosorbent assay (ELISA. The expression of proteins involved in cellular transdifferentiation was analyzed by western blot. Cell autophagy was measured by fluorescence microscopic analysis for acridine orange staining and transmission electron microscopy analysis. The autophagic inhibitors (3-MA, chloroquine, NH4Cl and bafilomycin A1 and β-catenin inhibitor JW74 were used to assess the effects of atorvastatin on autophagy and the involvement of β-catenin on cell calcification respectively. Furthermore, cell transfection was performed to overexpress β-catenin. Results: In VSMCs, atorvastatin significantly suppressed transforming growth factor-β1 (TGF-β1-stimulated calcification, accompanied by the induction of autophagy. Downregulation of autophagy with autophagic inhibitors significantly suppressed the inhibitory effect of atorvastatin on cell calcification. Moreover, the beneficial effect of atorvastatin on calcification and autophagy was reversed by β-catenin overexpression. Conversely, JW74 supplement enhanced this effect. Conclusion: These data demonstrated that atorvastatin protect VSMC from TGF-β1-stimulated calcification by inducing autophagy through suppression of the β-catenin pathway, identifying autophagy induction might be a therapeutic strategy for use in vascular calcification.

  15. 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.

  16. 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.

  17. Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

    Institute of Scientific and Technical Information of China (English)

    Lei Zhou; Jun Zhao; Wangzhen Guo; Tianzhen Zhang

    2013-01-01

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi.However,the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood.Here,we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes,VdATG8 and VdATG12,by means of targeted gene replacement and complementadon.Transformation of a cotton-infecting Verticilliun dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 106 conidia.V.dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production.Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants,compared with the wildtype and gene complemented strains.Surprisingly,in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants.These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V.dahliae.

  18. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    Science.gov (United States)

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  19. Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling.

    Science.gov (United States)

    Gong, F; Peng, X; Sang, Y; Qiu, M; Luo, C; He, Z; Zhao, X; Tong, A

    2013-11-07

    Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.

  20. N-Acetyl Cysteine Protects against Methamphetamine-Induced Dopaminergic Neurodegeneration via Modulation of Redox Status and Autophagy in Dopaminergic Cells

    Directory of Open Access Journals (Sweden)

    Prashanth Chandramani Shivalingappa

    2012-01-01

    Full Text Available Methamphetamine- (MA- induced neurotoxicity is associated with mitochondrial dysfunction and enhanced oxidative stress. Our previous study demonstrated that MA induces autophagy in a dopaminergic neuronal cell model (N27 cells. The cellular mechanisms underlying MA-induced autophagy and apoptosis remain poorly characterized. In the present study we sought to investigate the importance of GSH redox status in MA-induced neurotoxicity using a thiol antioxidant, N-acetylcysteine (NAC. Morphological and biochemical analysis revealed that MA-induced autophagy in N27 dopaminergic cells was associated with pronounced depletion of GSH levels. Moreover, pretreatment with NAC reduced MA-induced GSH depletion and autophagy, while depletion of GSH using L-buthionine sulfoximine (L-BSO enhanced autophagy. Furthermore, treatment with NAC significantly attenuated MA-induced apoptotic cell death as well as oxidative stress markers, namely, 3-nitrotyrosine (3-NT and 4-hydroxynonenal (4-HNE. Together, these results suggest that NAC exhibits significant protective effects against MA-induced dopaminergic cell death, presumably via modulation of the GSH level and autophagy. Collectively, our data provide mechanistic insights into the role of cellular GSH redox status in MA-induced autophagy and apoptotic cell death, and additional studies are needed to determine the therapeutic effectiveness of cellular redox modifiers in attenuating dopaminergic neurodegeneration in vivo.

  1. 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...

  2. Protective effects of crude garlic by reducing iron-mediated oxidative stress, proliferation and autophagy in rats.

    Science.gov (United States)

    Nahdi, Afef; Hammami, Imen; Kouidhi, Wided; Chargui, Abderrahman; Ben Ammar, Awatef; Hamdaoui, Mohamed Hédi; El May, Ahmed; El May, Michèle

    2010-10-01

    The impact of garlic, known for its antioxidant activities, on iron metabolism has been poorly investigated. The aim of this work was to study the effect of crude garlic pre-treatment on iron-mediated lipid peroxidation, proliferation and autophagy for 5 weeks. Rats were fed distilled water or garlic solution (1 g/kg body weight) by gavage for the first 3 weeks as pre-treatment and received a basal diet supplemented or not with ferrous sulfate (650 mg Fe/kg diet) for the last 2 weeks of treatment. Immunohistochemistry labeling and ultrastuctural observations were used to evaluate the iron deleterious effects in the liver. Iron supplementation induced cell proliferation predominantly in non parenchymal cells comparing to hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggers autophagy as evidenced by the formation of autophagic vesicles detected by LC3-II staining. It also induced morphologic alterations of the mitochondrial membranes due to increased lipid peroxidation as shown by elevated iron and malondialdehyde concentrations in serum and tissues. Garlic pre-treatment reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and by enhancing the status of antioxidants. In addition, garlic reduced the iron-mediated cell proliferation and autophagy by lowering iron storage in the liver and protected mitochondrial membrane. Based on these results, garlic treatment significantly prevented iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

  3. Celastrol protects human neuroblastoma SH-SY5Y cells from rotenone-induced injury through induction of autophagy.

    Science.gov (United States)

    Deng, Yong-Ning; Shi, Jie; Liu, Jie; Qu, Qiu-Min

    2013-07-01

    Celastrol, an active component found in the Chinese herb tripterygium wilfordii has been identified as a neuroprotective agent for neurodegenerative diseases including Parkinson's disease (PD) through unknown mechanism. Celastrol can induce autophagy, which plays a neuroprotective role in PD. We tested the protective effect of celastrol on rotenone-induced injury and investigated the underlying mechanism using human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were treated with celastrol before rotenone exposure. The cells survival, apoptosis, accumulation of α-synuclein, oxidative stress and mitochondrial function, and autophagy production were analyzed. We found celastrol (500 nM) pre-treatment enhanced cell viability (by 28.99%, P<0.001), decreased cell apoptosis (by 54.38%, P<0.001), increased SOD and GSH (by 120.53% and 90.46%, P<0.01), reduced accumulation of α-synuclein (by 35.93%, P<0.001) and ROS generation (by 33.99%, P<0.001), preserved MMP (33.93±3.62%, vs. 15.10±0.71% of JC-1 monomer, P<0.001) and reduced the level of cytochrome C in cytosol (by 45.57%, P<0.001) in rotenone treated SH-SY5Y cells. Moreover, celastrol increased LC3-II/LC3 I ratio by 60.92% (P<0.001), indicating that celastrol activated autophagic pathways. Inhibiting autophagy by 3-methyladenine (3-MA) abolished the protective effects of celastrol. Our results suggested that celastrol protects SH-SY5Y cells from rotenone induced injuries and autophagic pathway is involved in celastrol neuroprotective effects.

  4. Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Arwa S Kathiria

    Full Text Available INTRODUCTION: Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα, both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB, which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells. METHODS: We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine. RESULTS: TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability. CONCLUSIONS: Decreased PHB levels coupled with dysfunctional

  5. Altered gene expression and repressed markers of autophagy in skeletal muscle of insulin resistant patients with type 2 diabetes

    Science.gov (United States)

    Møller, Andreas Buch; Kampmann, Ulla; Hedegaard, Jakob; Thorsen, Kasper; Nordentoft, Iver; Vendelbo, Mikkel Holm; Møller, Niels; Jessen, Niels

    2017-01-01

    This case-control study was designed to investigate the gene expression profile in skeletal muscle from severely insulin resistant patients with long-standing type 2 diabetes (T2D), and to determine associated signaling pathways. Gene expression profiles were examined by whole transcriptome, strand-specific RNA-sequencing and associated signaling was determined by western blot. We identified 117 differentially expressed gene transcripts. Ingenuity Pathway Analysis related these differences to abnormal muscle morphology and mitochondrial dysfunction. Despite a ~5-fold difference in plasma insulin, we did not observe any difference in phosphorylation of AKT or AS160, although other insulin-sensitive cascades, as mTOR/4EBP1, had retained their sensitivity. Autophagy-related gene (ATG14, RB1CC1/FIP200, GABARAPL1, SQSTM1/p62, and WIPI1) and protein (LC3BII, SQSTM1/p62 and ATG5) expression were decreased in skeletal muscle from the patients, and this was associated with a trend to increased phosphorylation of the insulin-sensitive regulatory transcription factor FOXO3a. These data show that gene expression is highly altered and related to mitochondrial dysfunction and abnormal morphology in skeletal muscle from severely insulin resistant patients with T2D, and that this is associated with decreased expression of autophagy-related genes and proteins. We speculate that prolonged treatment with high doses of insulin may suppress autophagy thereby generating a vicious cycle maintaining insulin resistance. PMID:28252104

  6. Novel incretin analogues improve autophagy and protect from mitochondrial stress induced by rotenone in SH-SY5Y cells.

    Science.gov (United States)

    Jalewa, Jaishree; Sharma, Mohit Kumar; Hölscher, Christian

    2016-10-01

    Currently, there is no viable treatment available for Parkinson's disease (PD) that stops or reverses disease progression. Interestingly, studies testing the glucagon-like-peptide-1 (GLP-1) mimetic Exendin-4 have shown neuroprotective/neurorestorative properties in pre-clinical tests and in a pilot clinical study of PD. Incretin analogues were originally developed to treat type 2 diabetes and several are currently on the market. In this study, we tested novel incretin analogues on the dopaminergic SH-SY5Y neuroblastoma cells against a toxic mitochondrial complex I inhibitor, Rotenone. Here, we investigate for the first time the effects of six different incretin receptor agonists - Liraglutide, D-Ser2-Oxyntomodulin, a GLP-1/GIP Dual receptor agonist, dAla(2)-GIP-GluPal, Val(8)GLP-1-GluPal and exendin-4. Post-treatment with doses of 1, 10 or 100 nM of incretin analogues for 12 h increased the survival of SH-SY5Y cells treated with 1 μM Rotenone for 12 h. Furthermore, we studied the post-treatment effect of 100 nM incretin analogues against 1 μM Rotenone stress on apoptosis, mitochondrial stress and autophagy markers. We found significant protective effects of the analogues against Rotenone stress on cell survival and on mitochondrial and autophagy-associated markers. The novel GLP-1/GIP Dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. Using the Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we further show that the neuroprotective effects are partially PI3K-independent. Our data suggest that the neuroprotective properties exhibited by incretin analogues against Rotenone stress involve enhanced autophagy, increased Akt-mediated cell survival and amelioration of mitochondrial dysfunction. These mechanisms can explain the neuroprotective effects of incretin analogues reported in clinical trials. GLP-1, GIP and dual incretin receptor agonists showed protective effects in SH-SY5Y cells

  7. 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.

  8. Autophagy and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha kinase (eIF2α) pathway protect ovarian cancer cells from metformin-induced apoptosis.

    Science.gov (United States)

    Moon, Hee-Sun; Kim, Boyun; Gwak, HyeRan; Suh, Dong Hoon; Song, Yong Sang

    2016-04-01

    Metformin, an oral biguanide for the treatment of type II diabetes, has been shown to have anticancer effects in ovarian cancer. Energy starvation induced by metformin causes endoplasmic reticulum stress-mediated unfolded protein response (UPR) and autophagy. UPR and autophagy act as a survival or death mechanism in cells. In this study, we observed that metformin-induced apoptosis was relieved by autophagy and the PERK/eIF2α pathway in ovarian cancer cells, but not in peripheral blood mononuclear cells (PBMC) or 'normal' ovarian surface epithelial cells (OSE). Increased PARP cleavage and increased LC3B-II with ATG5-ATG12 complex suggested the induction of apoptosis and autophagy, respectively, in metformin-treated ovarian cancer cells. Accumulation of acidic vacuoles in the cytoplasm and downregulation of p62 further supported late-stage autophagy. Interestingly, metformin induced interdependent activation between autophagy and the UPR, especially the PERK/eIF2α pathway. Inhibition of autophagy-induced PERK inhibition, and vice versa, were demonstrated using small molecular inhibitors (PERK inhibitor I, GSK2606414; autophagy inhibitor, 3-MA, and BafA1). Moreover, autophagy and PERK activation protected ovarian cancer cells against metformin-induced apoptosis. Metformin treatment in the presence of inhibitors of PERK and autophagy, however, had no cytotoxic effects on OSE or PBMC. In conclusion, these results suggest that inhibition of autophagy and PERK can enhance the selective anticancer effects of metformin on ovarian cancer cells. © 2015 Wiley Periodicals, Inc.

  9. Induction of autophagy by salidroside through the AMPK-mTOR pathway protects vascular endothelial cells from oxidative stress-induced apoptosis.

    Science.gov (United States)

    Zheng, Xiang-Tao; Wu, Zi-Heng; Wei, Ye; Dai, Ju-Ji; Yu, Guan-Feng; Yuan, FengLai; Ye, Le-Chi

    2017-01-01

    Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H2O2). The results indicated that salidroside exerted cytoprotective effects in an H2O2-induced HUVEC injury model and suppressed H2O2-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.

  10. Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger.

    Science.gov (United States)

    Nitsche, Benjamin M; Burggraaf-van Welzen, Anne-Marie; Lamers, Gerda; Meyer, Vera; Ram, Arthur F J

    2013-09-01

    Autophagy is a well-conserved catabolic process constitutively active in eukaryotes that is involved in maintaining cellular homeostasis by the targeting of cytoplasmic content and organelles to vacuoles. Autophagy is strongly induced by the limitation of nutrients including carbon, nitrogen, and oxygen and is clearly associated with cell death. It has been demonstrated that the accumulation of empty hyphal compartments and cryptic growth in carbon-starved submerged cultures of the filamentous fungus Aspergillus niger is accompanied by a joint transcriptional induction of autophagy genes. This study examines the role of autophagy by deleting the atg1, atg8, and atg17 orthologs in A. niger and phenotypically analyzing the deletion mutants in surface and submerged cultures. The results indicate that atg1 and atg8 are essential for efficient autophagy, whereas deletion of atg17 has little to no effect on autophagy in A. niger. Depending on the kind of oxidative stress confronted with, autophagy deficiency renders A. niger either more resistant (menadione) or more sensitive (H2O2) to oxidative stress. Fluorescence microscopy showed that mitochondrial turnover upon carbon depletion in submerged cultures is severely blocked in autophagy-impaired A. niger mutants. Furthermore, automated image analysis demonstrated that autophagy promotes survival in maintained carbon-starved cultures of A. niger. Taken together, the results suggest that besides its function in nutrient recycling, autophagy plays important roles in physiological adaptation by organelle turnover and protection against cell death upon carbon depletion in submerged cultures.

  11. Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

    Science.gov (United States)

    Koenig, Ulrich; Fobker, Manfred; Lengauer, Barbara; Brandstetter, Marlene; Resch, Guenter P; Gröger, Marion; Plenz, Gabriele; Pammer, Johannes; Barresi, Caterina; Hartmann, Christine; Rossiter, Heidemarie

    2015-01-01

    The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to

  12. FGFR antagonist induces protective autophagy in FGFR1-amplified breast cancer cell.

    Science.gov (United States)

    Chen, Yi; Xie, Xiaoyan; Li, Xinyi; Wang, Peiqi; Jing, Qian; Yue, Jiaqi; Liu, Yang; Cheng, Zhong; Li, Jingyi; Song, Haixing; Li, Guoyu; Liu, Rui; Wang, Jinhui

    2016-05-20

    Breast cancer, representing approximately 30% of all gynecological cancer cases diagnosed yearly, is a leading cause of cancer-related mortality for women. Amplification of FGFR1 is frequently observed in breast cancers and is associated with poor prognosis. Though FGFRs have long been considered as anti-cancer drug targets, and a cluster of FGFR antagonists are currently under clinical trials, the precise cellular responses under the treatment of FGFR antagonists remains unclear. Here, we show that PD166866, an FGFR1-selective inhibitor, inhibits proliferation and triggers anoikis in FGFR1-amplified breast cancer cell lines. Notably, we demonstrate that PD166866 induces autophagy in FGFR1-amplified breast cancer cell lines, while blockage of autophagy by Atg5 knockdown further enhances the anti-proliferative activities of PD166866. Moreover, mechanistic study reveals that PD166866 induces autophagy through repressing Akt/mTOR signaling pathway. Together, the present study provides new insights into the molecular mechanisms underlying the anti-tumor activities of FGFR antagonists, and may further assist the FGFRs-based drug discovery.

  13. Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy

    Science.gov (United States)

    Huuskonen, Mikko T.; Loppi, Sanna; Dhungana, Hiramani; Keksa-Goldsteine, Velta; Lemarchant, Sighild; Korhonen, Paula; Wojciechowski, Sara; Pollari, Eveliina; Valonen, Piia; Koponen, Juho; Takashima, Akihiko; Landreth, Gary; Goldsteins, Gundars; Malm, Tarja; Koistinaho, Jari; Kanninen, Katja M.

    2016-01-01

    Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene. PMID:27624652

  14. Sulforaphane protects against rotenone-induced neurotoxicity in vivo: Involvement of the mTOR, Nrf2, and autophagy pathways

    Science.gov (United States)

    Zhou, Qian; Chen, Bin; Wang, Xindong; Wu, Lixin; Yang, Yang; Cheng, Xiaolan; Hu, Zhengli; Cai, Xueting; Yang, Jie; Sun, Xiaoyan; Lu, Wuguang; Yan, Huaijiang; Chen, Jiao; Ye, Juan; Shen, Jianping; Cao, Peng

    2016-01-01

    Sulforaphane, a naturally occurring compound found in cruciferous vegetables, has been shown to be neuroprotective in several neurological disorders. In this study, we sought to investigate the potential protective effects and associated molecular mechanisms of sulforaphane in an in vivo Parkinson’s disease (PD) model, based on rotenone-mediated neurotoxicity. Our results showed that sulforaphane inhibited rotenone-induced locomotor activity deficiency and dopaminergic neuronal loss. Additionally, sulforaphane treatment inhibited the rotenone-induced reactive oxygen species production, malondialdehyde (MDA) accumulation, and resulted in an increased level of total glutathione and reduced glutathione (GSH): oxidized glutathione (GSSG) in the brain. Western blot analysis illustrated that sulforaphane increased the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase (NQO1), the latter two of which are anti-oxidative enzymes. Moreover, sulforaphane treatment significantly attenuated rotenone-inhibited mTOR-mediated p70S6K and 4E-BP1 signalling pathway, as well as neuronal apoptosis. In addition, sulforaphane rescued rotenone-inhibited autophagy, as detected by LC3-II. Collectively, these findings demonstrated that sulforaphane exert neuroprotective effect involving Nrf2-dependent reductions in oxidative stress, mTOR-dependent inhibition of neuronal apoptosis, and the restoration of normal autophagy. Sulforaphane appears to be a promising compound with neuroprotective properties that may play an important role in preventing PD. PMID:27553905

  15. The role of autophagy in the intracellular survival of Campylobacter concisus

    Directory of Open Access Journals (Sweden)

    Jose A. Burgos-Portugal

    2014-01-01

    Full Text Available Campylobacter concisus is an emerging pathogen that has been associated with gastrointestinal diseases. Given the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria, we investigated the role of autophagy in C. concisus intracellular survival. Gentamicin protection assays were employed to assess intracellular levels of C. concisus within Caco-2 cells, following autophagy induction and inhibition. To assess the interaction between C. concisus and autophagosomes, confocal microscopy, scanning electron microscopy, and transmission electron microscopy were employed. Expression levels of 84 genes involved in the autophagy process were measured using qPCR. Autophagy inhibition resulted in two- to four-fold increases in intracellular levels of C. concisus within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. C. concisus strains with low intracellular survival levels showed a dramatic increase in these levels upon autophagy inhibition. Confocal microscopy showed co-localization of the bacterium with autophagosomes, while transmission electron microscopy identified intracellular bacteria persisting within autophagic vesicles. Further, qPCR showed that following infection, 13 genes involved in the autophagy process were significantly regulated, and a further five showed borderline results, with an overall indication towards a dampening effect exerted by the bacterium on this process. Our data collectively indicates that while autophagy is important for the clearance of C. concisus, some strains may manipulate this process to benefit their intracellular survival.

  16. Autophagy in Hepatic Fibrosis

    Directory of Open Access Journals (Sweden)

    Yang Song

    2014-01-01

    Full Text Available Hepatic fibrosis is a leading cause of morbidity and mortality worldwide. Hepatic fibrosis is usually associated with chronic liver diseases caused by infection, drugs, metabolic disorders, or autoimmune imbalances. Effective clinical therapies are still lacking. Autophagy is a cellular process that degrades damaged organelles or protein aggregation, which participates in many pathological processes including liver diseases. Autophagy participates in hepatic fibrosis by activating hepatic stellate cells and may participate as well through influencing other fibrogenic cells. Besides that, autophagy can induce some liver diseases to develop while it may play a protective role in hepatocellular abnormal aggregates related liver diseases and reduces fibrosis. With a better understanding of the potential effects of autophagy on hepatic fibrosis, targeting autophagy might be a novel therapeutic strategy for hepatic fibrosis in the near future.

  17. Autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation

    Directory of Open Access Journals (Sweden)

    Bullon Pedro

    2012-10-01

    Full Text Available Abstract Background Periodontitis, the most prevalent chronic inflammatory disease, has been related to cardiovascular diseases. Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. The aim of this research was to study the role of autophagy in peripheral blood mononuclear cells from patients with periodontitis and gingival fibroblasts treated with a lipopolysaccharide of Porphyromonas gingivalis. Autophagy-dependent mechanisms have been proposed in the pathogenesis of inflammatory disorders and in other diseases related to periodontitis, such as cardiovascular disease and diabetes. Thus it is important to study the role of autophagy in the pathophysiology of periodontitis. Methods Peripheral blood mononuclear cells from patients with periodontitis (n = 38 and without periodontitis (n = 20 were used to study autophagy. To investigate the mechanism of autophagy, we evaluated the influence of a lipopolysaccharide from P. gingivalis in human gingival fibroblasts, and autophagy was monitored morphologically and biochemically. Autophagosomes were observed by immunofluorescence and electron microscopy. Results We found increased levels of autophagy gene expression and high levels of mitochondrial reactive oxygen species production in peripheral blood mononuclear cells from patients with periodontitis compared with controls. A significantly positive correlation between both was observed. In human gingival fibroblasts treated with lipopolysaccharide from P. gingivalis, there was an increase of protein and transcript of autophagy-related protein 12 (ATG12 and microtubule-associated protein 1 light chain 3 alpha LC3. A reduction of mitochondrial reactive oxygen species induced a decrease in autophagy whereas inhibition of autophagy in infected cells increased apoptosis, showing the protective role of autophagy. Conclusion Results from the present study suggest that autophagy

  18. A novel protective mechanism for mitochondrial aldehyde dehydrogenase (ALDH2) in type i diabetes-induced cardiac dysfunction: role of AMPK-regulated autophagy.

    Science.gov (United States)

    Guo, Yuli; Yu, Wenjun; Sun, Dongdong; Wang, Jiaxing; Li, Congye; Zhang, Rongqing; Babcock, Sara A; Li, Yan; Liu, Min; Ma, Meijuan; Shen, Mingzhi; Zeng, Chao; Li, Na; He, Wei; Zou, Qian; Zhang, Yingmei; Wang, Haichang

    2015-02-01

    Mitochondrial aldehyde dehydrogenase (ALDH2) is known to offer myocardial protection against stress conditions including ischemia-reperfusion injury, alcoholism and diabetes mellitus although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on diabetes-induced myocardial injury with a focus on autophagy. Wild-type FVB and ALDH2 transgenic mice were challenged with streptozotozin (STZ, 200mg/kg, i.p.) for 3months to induce experimental diabetic cardiomyopathy. Diabetes triggered cardiac remodeling and contractile dysfunction as evidenced by cardiac hypertrophy, decreased cell shortening and prolonged relengthening duration, the effects of which were mitigated by ALDH2. Lectin staining displayed that diabetes promoted cardiac hypertrophy, the effect of which was alleviated by ALDH2. Western blot analysis revealed dampened autophagy protein markers including LC3B ratio and Atg7 along with upregulated p62 following experimental diabetes, the effect of which was reconciled by ALDH2. Phosphorylation level of AMPK was decreased and its downstream signaling molecule FOXO3a was upregulated in both diabetic cardiac tissue and in H9C2 cells with high glucose exposure. All these effect were partly abolished by ALDH2 overexpression and ALDH2 agonist Alda1. High glucose challenge dampened autophagy in H9C2 cells as evidenced by enhanced p62 levels and decreased levels of Atg7 and LC3B, the effect of which was alleviated by the ALDH2 activator Alda-1. High glucose-induced cell death and apoptosis were reversed by Alda-1. The autophagy inhibitor 3-MA and the AMPK inhibitor compound C mitigated Alda-1-offered beneficial effect whereas the autophagy inducer rapamycin mimicked or exacerbated high glucose-induced cell injury. Moreover, compound C nullified Alda-1-induced protection against STZ-induced changes in autophagy and function. Our results suggested that ALDH2 protects against diabetes-induced myocardial dysfunction possibly through an

  19. Relationship between autophagy-related genes Beclin-1 and MAP1LC3 expression and biological characteristics of oral cancer

    Institute of Scientific and Technical Information of China (English)

    Xiao-Dong Li; Xiao-Chen Sun; Xin-Mei Li; Jia-Wei Gu

    2016-01-01

    Objective:To study the relationship between autophagy-related genes Beclin-1 and MAP1LC3 expression and biological characteristics of oral cancer. Methods:Oral cancer tissues and precancerous tissues were collected to detect mRNA expression levels of Beclin-1 and MAP1LC3;tongue cancer cell lines CTST-2 and primary epithelial cells of normal buccal mucosa were cultured to detect mRNA expression levels of autophagy marker molecues (Beclin-1 and MAP1LC3), pro-apoptosis genes (P53 and Caspase-3) and anti-apoptosis genes (Survivin, Bcl-2 and Bmi-1). Results:mRNA contents of Beclin-1 and MAP1LC3 in tongue cancer, buccal mucosa cancer, gingival cancer and mouth floor cancer tissues were significantly lower than those in corresponding precancerous tissues; mRNA contents of Beclin-1 and MAP1LC3 in tongue cancer cells CTST-2 were lower than those in normal mucosal cells;mRNA contents of P53 and Caspase-3 in tongue cancer cells CTST-2 were lower than those in normal mucosal cells and positively correlated with mRNA contents of Beclin-1 and MAP1LC3; mRNA contents of survivin, Bcl-2 and Bmi-1 in CTST-2 were higher than those in normal mucosal cells and negatively correlated with mRNA contents of Beclin-1 and MAP1LC3. Conclusion:Expression levels of autophagy-related genes Beclin-1 and MAP1LC3 abnormally reduce in oral cancer and have significant correlation with the expression of pro-apoptosis genes and anti-apoptosis genes of cancer cells.

  20. 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).

  1. A role for autophagy in the extension of lifespan by dietary restriction in C. elegans.

    Directory of Open Access Journals (Sweden)

    Malene Hansen

    2008-02-01

    Full Text Available In many organisms, dietary restriction appears to extend lifespan, at least in part, by down-regulating the nutrient-sensor TOR (Target Of Rapamycin. TOR inhibition elicits autophagy, the large-scale recycling of cytoplasmic macromolecules and organelles. In this study, we asked whether autophagy might contribute to the lifespan extension induced by dietary restriction in C. elegans. We find that dietary restriction and TOR inhibition produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan. The longevity response to dietary restriction in C. elegans requires the PHA-4 transcription factor. We find that the autophagic response to dietary restriction also requires PHA-4 activity, indicating that autophagy is a transcriptionally regulated response to food limitation. In spite of the rejuvenating effect that autophagy is predicted to have on cells, our findings suggest that autophagy is not sufficient to extend lifespan. Long-lived daf-2 insulin/IGF-1 receptor mutants require both autophagy and the transcription factor DAF-16/FOXO for their longevity, but we find that autophagy takes place in the absence of DAF-16. Perhaps autophagy is not sufficient for lifespan extension because although it provides raw material for new macromolecular synthesis, DAF-16/FOXO must program the cells to recycle this raw material into cell-protective longevity proteins.

  2. Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

    Science.gov (United States)

    Li, Yuan-Yuan; Wang, Ting; Gao, Song; Xu, Guang-Mei; Niu, Hua; Huang, Rui; Wu, Shu-Yan

    2016-02-01

    Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

  3. Autophagy and apoptosis-related genes in chronic liver disease and hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Kotsafti Andromachi

    2012-08-01

    Full Text Available Abstract Background Dysregulation of autophagy is important in the pathogenesis of many diseases, including cancer. Several aspects of the biological role of autophagy are however still unclear and the relationship between apoptosis and autophagy, particularly in the liver has yet to be thoroughly explored. In this study we evaluated the expression of Beclin 1 (one of the main autophagocytic agents, which bridges autophagy, apoptosis and both differentiation, and both pro- (Bad, Bax and anti-apoptotic (Bcl-2, Bcl-xL factors in liver samples from patients with different stages of liver disease. Methods The study concerned 93 patients from 49 cases of chronic hepatitis (CH (30 HCV and 19 HBV-related, 13 of cirrhosis (CIRR (10 HCV and 3 HBV-related, 21 of hepatocellular carcinoma (both HCC and peritumoral tissues [PHCC], and 10 controls (CONTR. Real-time PCR and Western blotting were used to measure mRNA and protein expression levels. Results Beclin 1 mRNA levels were lower in HCC than in CH (P = 0.010 or CIRR (P = 0.011, and so were the Bcl-xL transcripts (P  Conclusions High Beclin 1, Bcl-xL and Bad levels in CH and CIRR tissues suggest an interaction between autophagy and apoptosis in the early and intermediate stages of viral hepatitis. In HCC these processes seem to be downregulated, probably enabling the survival and growth of neoplastic hepatocytes.

  4. Protective effect of astaxanthin on liver fibrosis through modulation of TGF-β1 expression and autophagy.

    Science.gov (United States)

    Shen, Miao; Chen, Kan; Lu, Jie; Cheng, Ping; Xu, Ling; Dai, Weiqi; Wang, Fan; He, Lei; Zhang, Yan; Chengfen, Wang; Li, Jingjing; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Zhou, Yingqun; Guo, Chuanyong

    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, 40, and 80 mg/kg). Pathological results indicated that astaxanthin significantly improved the pathological lesions of liver fibrosis. The levels of alanine aminotransferase aspartate aminotransferase and hydroxyproline were also significantly decreased by astaxanthin. The same results were confirmed in bile duct liagtion, (BDL) model. In addition, astaxanthin inhibited hepatic stellate cells (HSCs) activation and formation of extracellular matrix (ECM) by decreasing the expression of NF-κB and TGF-β1 and maintaining the balance between MMP2 and TIMP1. In addition, astaxanthin reduced energy production in HSCs by downregulating the level of autophagy. These results were simultaneously confirmed in vivo and in vitro. In conclusion, our study showed that 80 mg/kg astaxanthin had a significant protective effect on liver fibrosis by suppressing multiple profibrogenic factors.

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

    Directory of Open Access Journals (Sweden)

    Miao Shen

    2014-01-01

    Full Text Available 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, 40, and 80 mg/kg. Pathological results indicated that astaxanthin significantly improved the pathological lesions of liver fibrosis. The levels of alanine aminotransferase aspartate aminotransferase and hydroxyproline were also significantly decreased by astaxanthin. The same results were confirmed in bile duct liagtion, (BDL model. In addition, astaxanthin inhibited hepatic stellate cells (HSCs activation and formation of extracellular matrix (ECM by decreasing the expression of NF-κB and TGF-β1 and maintaining the balance between MMP2 and TIMP1. In addition, astaxanthin reduced energy production in HSCs by downregulating the level of autophagy. These results were simultaneously confirmed in vivo and in vitro. In conclusion, our study showed that 80 mg/kg astaxanthin had a significant protective effect on liver fibrosis by suppressing multiple profibrogenic factors.

  6. Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation.

    Science.gov (United States)

    Mortensen, Monika; Watson, Alexander Scarth; Simon, Anna Katharina

    2011-09-01

    The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).

  7. 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.

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

    DEFF Research Database (Denmark)

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V

    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 ...

  9. 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.

  10. The autophagy gene Wdr45/Wipi4 regulates learning and memory function and axonal homeostasis.

    Science.gov (United States)

    Zhao, Yan G; Sun, Le; Miao, Guangyan; Ji, Cuicui; Zhao, Hongyu; Sun, Huayu; Miao, Lin; Yoshii, Saori R; Mizushima, Noboru; Wang, Xiaoqun; Zhang, Hong

    2015-01-01

    WDR45/WIPI4, encoding a WD40 repeat-containing PtdIns(3)P binding protein, is essential for the basal autophagy pathway. Mutations in WDR45 cause the neurodegenerative disease β-propeller protein-associated neurodegeneration (BPAN), a subtype of NBIA. We generated CNS-specific Wdr45 knockout mice, which exhibit poor motor coordination, greatly impaired learning and memory, and extensive axon swelling with numerous axon spheroids. Autophagic flux is defective and SQSTM1 (sequestosome-1)/p62 and ubiquitin-positive protein aggregates accumulate in neurons and swollen axons. Nes-Wdr45(fl/Y) mice recapitulate some hallmarks of BPAN, including cognitive impairment and defective axonal homeostasis, providing a model for revealing the disease pathogenesis of BPAN and also for investigating the possible role of autophagy in axon maintenance.

  11. 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

    2016-06-09

    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.

  12. Investigating regulatory signatures of human autophagy related gene 5 (ATG5 through functional in silico analysis

    Directory of Open Access Journals (Sweden)

    Avni Vij

    2016-09-01

    Full Text Available Autophagy is an essential, homeostatic process which removes damaged cellular proteins and organelles for cellular renewal. ATG5, a part of E3 ubiquitin ligase-like complex (Atg12-Atg5/Atg16L1, is a key regulator involved in autophagosome formation - a crucial phase of autophagy. In this study, we used different in silico methods for comprehensive analysis of ATG5 to investigate its less explored regulatory activity. We have predicted various physico-chemical parameters and two possible transmembrane models that helped in exposing its functional regions. Twenty four PTM sites and 44 TFBS were identified which could be targeted to modulate the autophagy pathway. Furthermore, LD analysis identified 3 blocks of genotyped SNPs and 2 deleterious nsSNPs that may have damaging impact on protein function and thus could be employed for carrying genome-wide association studies. In conclusion, the information obtained in this study could be helpful for better understanding of regulatory roles of ATG5 and provides a base for its implication in population-based studies.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Chun-Yan [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi' an 710032 (China); Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Guo, Xiao-Zhong, E-mail: guoxiaozhong1962@163.com [Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Wang, Hua, E-mail: wanghua@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2011-01-21

    Research highlights: {yields} We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. {yields} Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. {yields} 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.

  14. Autophagy in sepsis: Degradation into exhaustion?

    Science.gov (United States)

    Ho, Jeffery; Yu, Jun; Wong, Sunny H; Zhang, Lin; Liu, Xiaodong; Wong, Wai T; Leung, Czarina C H; Choi, Gordon; Wang, Maggie H T; Gin, Tony; Chan, Matthew T V; Wu, William K K

    2016-07-01

    Autophagy is one of the innate immune defense mechanisms against microbial challenges. Previous in vitro and in vivo models of sepsis demonstrated that autophagy was activated initially in sepsis, followed by a subsequent phase of impairment. Autophagy modulation appears to be protective against multiple organ injuries in these murine sepsis models. This is achieved in part by preventing apoptosis, maintaining a balance between the productions of pro- and anti-inflammatory cytokines, and preserving mitochondrial functions. This article aims to discuss the role of autophagy in sepsis and the therapeutic potential of autophagy enhancers.

  15. 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.

  16. Autophagy in cancer: good, bad, or both?

    Science.gov (United States)

    Hippert, Melanie M; O'Toole, Patrick S; Thorburn, Andrew

    2006-10-01

    Autophagy has been recognized as an important cellular process for at least 50 years; however, it is only with the recent identification of key regulators of autophagy (Atg genes) that we have begun a mechanistic exploration of its importance in cancer. 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. However, the role of autophagy in these processes is complicated and may, depending on the circumstances, have diametrically opposite consequences for the tumor. In this article, we discuss recent discoveries regarding autophagy in cancer.

  17. Inducing autophagy

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  18. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children

    Directory of Open Access Journals (Sweden)

    Sergio Comincini

    2017-02-01

    Full Text Available Celiac disease (CD is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1 and their regulatory validated miRNAs (miR-17 and miR-30a, respectively were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann–Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy

  19. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children

    Science.gov (United States)

    Comincini, Sergio; Manai, Federico; Meazza, Cristina; Pagani, Sara; Martinelli, Carolina; Pasqua, Noemi; Pelizzo, Gloria; Biggiogera, Marco; Bozzola, Mauro

    2017-01-01

    Celiac disease (CD) is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann–Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process within a

  20. 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.

  1. The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800MHz radiofrequency electromagnetic radiation.

    Science.gov (United States)

    Liu, Kaijun; Zhang, Guowei; Wang, Zhi; Liu, Yong; Dong, Jianyun; Dong, Xiaomei; Liu, Jinyi; Cao, Jia; Ao, Lin; Zhang, Shaoxiang

    2014-08-04

    The increasing exposure to radiofrequency (RF) radiation emitted from mobile phone use has raised public concern regarding the biological effects of RF exposure on the male reproductive system. Autophagy contributes to maintaining intracellular homeostasis under environmental stress. To clarify whether RF exposure could induce autophagy in the spermatocyte, mouse spermatocyte-derived cells (GC-2) were exposed to 1800MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rate (SAR) values of 1w/kg, 2w/kg or 4w/kg for 24h, respectively. The results indicated that the expression of LC3-II increased in a dose- and time-dependent manner with RF exposure, and showed a significant change at the SAR value of 4w/kg. The autophagosome formation and the occurrence of autophagy were further confirmed by GFP-LC3 transient transfection assay and transmission electron microscopy (TEM) analysis. Furthermore, the conversion of LC3-I to LC3-II was enhanced by co-treatment with Chloroquine (CQ), indicating autophagic flux could be enhanced by RF exposure. Intracellular ROS levels significantly increased in a dose- and time-dependent manner after cells were exposed to RF. Pretreatment with anti-oxidative NAC obviously decreased the conversion of LC3-I to LC3-II and attenuated the degradation of p62 induced by RF exposure. Meanwhile, phosphorylated extracellular-signal-regulated kinase (ERK) significantly increased after RF exposure at the SAR value of 2w/kg and 4w/kg. Moreover, we observed that RF exposure did not increase the percentage of apoptotic cells, but inhibition of autophagy could increase the percentage of apoptotic cells. These findings suggested that autophagy flux could be enhanced by 1800MHz GSM exposure (4w/kg), which is mediated by ROS generation. Autophagy may play an important role in preventing cells from apoptotic cell death under RF exposure stress.

  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. Autophagy in colorectal cancer:An important switch from physiology to pathology

    Institute of Scientific and Technical Information of China (English)

    Florin; Burada; Elena; Raluca; Nicoli; Marius; Eugen; Ciurea; Daniel; Constantin; Uscatu; Mihai; Ioana; Dan; Ionut; Gheonea

    2015-01-01

    Colorectal cancer(CRC) remains a leading cause of cancer death in both men and women worldwide.Among the factors and mechanisms that are involved in the multifactorial etiology of CRC,autophagy is an important transformational switch that occurs when a cell shifts from normal to malignant.In recent years,multiple hypotheses have been considered regarding the autophagy mechanisms that are involved in cancer.The currently accepted hypothesis is that autophagy has dual and contradictory roles in carcinogenesis,but the precise mechanisms leading to autophagy in cancer are not yet fully defined and seem to be context dependent.Autophagy is a surveillance mechanism used by normal cells that protects them from the transformation to malignancy by removing damaged organelles and aggregated proteins and by reducing reactive oxygen species,mitochondrial abnormalities and DNA damage.However,autophagy also supports tumor formation by promoting access to nutrients that are critical to the metabolism and growth of tumor cells and by inhibiting cellular death and increasing drug resistance.Autophagy studies in CRC have focused on several molecules,mainly microtubule-associated protein 1 light chain 3,beclin 1,and autophagy related 5,with conflicting results.Beneficial effects were observed for some agents that modulate autophagy in CRC either alone or,more often,in combination with other agents.More extensive studies are needed in the future to clarify the roles of autophagy-related genes and modulators in colorectal carcinogenesis,and to develop potential beneficial agents for the prognosis and treatment of CRC.

  4. 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 (PGR activation indicated by higher ratio of GR phosphorylation. Out of 17 autophagy-related genes determined, 8 was significantly (PGR binding to the promoter sequence of ATG3 and Per2. Moreover, MeDIP analysis demonstrated significant (PGR-mediated pathway. Our results provide a basis for future investigations into the intracellular regulatory network in response to folate deficiency.

  5. Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration.

    Science.gov (United States)

    Zhang, J; Bai, Y; Huang, L; Qi, Y; Zhang, Q; Li, S; Wu, Y; Li, X

    2015-11-12

    Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the

  6. Autophagy: An Exposing Therapeutic Target in Atherosclerosis.

    Science.gov (United States)

    Luo, Yun; Lu, Shan; Zhou, Ping; Ai, Qi-Di; Sun, Gui-Bo; Sun, Xiao-Bo

    2016-03-01

    Autophagy is an evolutionarily conserved catabolic process whereby the cytoplasmic contents of a cell are sequestered within autophagosomes through a lysosome-dependent pathway. Increasing evidence shows that this process is of great importance in a wide range of diseases, including atherosclerosis (AS). Autophagy can be modulated in advanced AS plaques by cytokines, reactive lipids, lipopolysaccharides, advanced glycation end products, and microRNAs. Autophagy exerts both protective and detrimental functions in vascular disorders. However, despite an increasing interest in autophagy, it remains an underestimated and overlooked phenomenon in AS. Therefore, the precise role of autophagy and its relationship with apoptosis need to be described. This review highlights recent findings on the autophagy activities and signaling pathways in endothelial cells, macrophages, and smooth muscle cells that are accompanied by apoptosis in AS. We conclude with recent studies on autophagy modulation as a new therapeutic approach to treat AS.

  7. Cloning, expression analysis, and RNA interference study of a HORMA domain containing autophagy-related gene 13 (ATG13) from the coleopteran beetle, Tenebrio molitor.

    Science.gov (United States)

    Lee, Jung Hee; Jo, Yong Hun; Patnaik, Bharat Bhusan; Park, Ki Beom; Tindwa, Hamisi; Seo, Gi Won; Chandrasekar, Raman; Lee, Yong Seok; Han, Yeon Soo

    2015-01-01

    Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships.

  8. 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.

  9. 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.

  10. Identification, molecular cloning and expression analysis of a HORMA domain containing Autophagy-related gene 13 (ATG13 from the coleopteran beetle, Tenebrio molitor

    Directory of Open Access Journals (Sweden)

    Jung Hee eLee

    2015-06-01

    Full Text Available Autophagy is a process that is necessary during starvation as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg proteins that manifest in the nucleation, elongation, and curving of autophagosome membrane. We isolated a homolog of an ATG13 gene from the transcriptome database of the larva of the mealworm beetle, Tenebrio molitor (designated as TmATG13. The sequence analysis showed that TmATG13 cDNA comprises of 1,176 bp open reading frame that encodes a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region, rich in regulatory phosphorylation sites. The N-terminal Atg13 domain show a HORMA (Hop1, Rev7, and Mad2 fold containing conserved amino acid residues across the Atg13 orthologs in insects. qRT-PCR revealed that TmATG13 was expressed ubiquitously in all the developmental stages of insect. TmATG13 mRNA expression was high in fat body and gut of the larval and adult stages of the insect. During ovary development and maturation, the TmATG13 transcripts showed high expression until six days of development, followed by a significant decline. The prospective functions mediated by TmAtg13 during autophagy will be clarified by further studies in the near future.

  11. Far-infrared protects vascular endothelial cells from advanced glycation end products-induced injury via PLZF-mediated autophagy in diabetic mice

    Science.gov (United States)

    Chen, Cheng-Hsien; Chen, Tso-Hsiao; Wu, Mei-Yi; Chou, Tz-Chong; Chen, Jia-Rung; Wei, Meng-Jun; Lee, San-Liang; Hong, Li-Yu; Zheng, Cai-Mei; Chiu, I-Jen; Lin, Yuh-Feng; Hsu, Ching-Min; Hsu, Yung-Ho

    2017-01-01

    The accumulation of advanced glycation end products (AGEs) in diabetic patients induces vascular endothelial injury. Promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor that can be activated by low-temperature far-infrared (FIR) irradiation to exert beneficial effects on the vascular endothelium. In the present study, we investigated the influence of FIR-induced PLZF activation on AGE-induced endothelial injury both in vitro and in vivo. FIR irradiation inhibited AGE-induced apoptosis in human umbilical vein endothelial cells (HUVECs). PLZF activation increased the expression of phosphatidylinositol-3 kinases (PI3K), which are important kinases in the autophagic signaling pathway. FIR-induced PLZF activation led to autophagy in HUVEC, which was mediated through the upregulation of PI3K. Immunofluorescence staining showed that AGEs were engulfed by HUVECs and localized to lysosomes. FIR-induced autophagy promoted AGEs degradation in HUVECs. In nicotinamide/streptozotocin-induced diabetic mice, FIR therapy reduced serum AGEs and AGEs deposition at the vascular endothelium. FIR therapy also reduced diabetes-induced inflammatory markers in the vascular endothelium and improved vascular endothelial function. These protective effects of FIR therapy were not found in PLZF-knockout mice. Our data suggest that FIR-induced PLZF activation in vascular endothelial cells protects the vascular endothelium in diabetic mice from AGE-induced injury. PMID:28071754

  12. 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.

  13. The role of STAT3 in autophagy.

    Science.gov (United States)

    You, Liangkun; Wang, Zhanggui; Li, Hongsen; Shou, Jiawei; Jing, Zhao; Xie, Jiansheng; Sui, Xinbing; Pan, Hongming; Han, Weidong

    2015-01-01

    Autophagy is an evolutionarily conserved process in eukaryotes that eliminates harmful components and maintains cellular homeostasis in response to a series of extracellular insults. However, these insults may trigger the downstream signaling of another prominent stress responsive pathway, the STAT3 signaling pathway, which has been implicated in multiple aspects of the autophagic process. Recent reports further indicate that different subcellular localization patterns of STAT3 affect autophagy in various ways. For example, nuclear STAT3 fine-tunes autophagy via the transcriptional regulation of several autophagy-related genes such as BCL2 family members, BECN1, PIK3C3, CTSB, CTSL, PIK3R1, HIF1A, BNIP3, and microRNAs with targets of autophagy modulators. Cytoplasmic STAT3 constitutively inhibits autophagy by sequestering EIF2AK2 as well as by interacting with other autophagy-related signaling molecules such as FOXO1 and FOXO3. Additionally, the mitochondrial translocation of STAT3 suppresses autophagy induced by oxidative stress and may effectively preserve mitochondria from being degraded by mitophagy. Understanding the role of STAT3 signaling in the regulation of autophagy may provide insight into the classic autophagy model and also into cancer therapy, especially for the emerging targeted therapy, because a series of targeted agents execute antitumor activities via blocking STAT3 signaling, which inevitably affects the autophagy pathway. Here, we review several of the representative studies and the current understanding in this particular field.

  14. Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: new implications for osteonecrosis treatment?

    Science.gov (United States)

    She, Chang; Zhu, Lun-qing; Zhen, Yun-fang; Wang, Xiao-dong; Dong, Qi-rong

    2014-01-01

    Elevated hydrogen peroxide (H2O2) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H2O2 stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H2O2-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H2O2-induced cell damage. These results confirmed that H2O2-activated AMPK is pro-cell survival. We observed that H2O2 induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H2O2-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H2O2 in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H2O2-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H2O2 in these cells. Based on these results, we concluded that H2O2-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.

  15. 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.

  16. Feedback regulation between autophagy and PKA.

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

  17. Feedback regulation between autophagy and PKA

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA. PMID:26046386

  18. 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

  19. 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.

  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. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

    Energy Technology Data Exchange (ETDEWEB)

    Ariyoshi, Kentaro [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei [Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Oshimura, Mitsuo [Chromosome Engineering Research Center (CERC), Tottori University, Nishicho 86, Yonago, Tottori 683-8503 (Japan); Yoshida, Mitsuaki A., E-mail: ariyoshi@hirosaki-u.ac.jp [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan)

    2016-08-15

    Highlights: • Clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. • Increased autophagy was observed in the artificially aneuploid clones. • Inhibition of autophagy resulted in increased genomic instability and DNA damage. • Intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones. - Abstract: Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

  2. 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

    2016-01-01

    ABSTRACT 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

  3. 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 discussion have been focused on the scope of protection (e.g. purpose-bound protection) and gene patents being subject to a specific DNA regime on patent rights. The Directive can be interpreted as favouring such a solution, but so far the European Commission has decided neither to support nor reject...

  4. Modulation of Apoptosis Pathways by Oxidative Stress and Autophagy in β Cells

    Directory of Open Access Journals (Sweden)

    Maorong Wang

    2012-01-01

    Full Text Available Human islets isolated for transplantation are exposed to multiple stresses including oxidative stress and hypoxia resulting in significant loss of functional β cell mass. In this study we examined the modulation of apoptosis pathway genes in islets exposed to hydrogen peroxide, peroxynitrite, hypoxia, and cytokines. We observed parallel induction of pro- and antiapoptotic pathways and identified several novel genes including BFAR, CARD8, BNIP3, and CIDE-A. As BNIP3 is an inducer of autophagy, we examined this pathway in MIN6 cells, a mouse beta cell line and in human islets. Culture of MIN6 cells under low serum conditions increased the levels of several proteins in autophagy pathway, including ATG4, Beclin 1, LAMP-2, and UVRAG. Amino acid deprivation led to induction of autophagy in human islets. Preconditioning of islets with inducers of autophagy protected them from hypoxia-induced apoptosis. However, induction of autophagy during hypoxia exacerbated apoptotic cell death. ER stress led to induction of autophagy and apoptosis in β cells. Overexpression of MnSOD, an enzyme that scavenges free radicals, resulted in protection of MIN6 cells from cytokine-induced apoptosis. Ceramide, a mediator of cytokine-induced injury, reduced the active phosphorylated form of Akt and downregulated the promoter activity of the antiapoptotic gene bcl-2. Furthermore, cytokine-stimulated JNK pathway downregulated the bcl-2 promoter activity which was reversed by preincubation with SP600125, a JNK inhibitor. Our findings suggest that β cell apoptosis by multiple stresses in islets isolated for transplantation is the result of orchestrated gene expression in apoptosis pathway.

  5. 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.

  6. 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.

  7. Autophagy and the nutritional signaling pathway

    Directory of Open Access Journals (Sweden)

    Long HE,Shabnam ESLAMFAM,Xi MA,Defa LI

    2016-09-01

    Full Text Available During their growth and development, animals adapt to tremendous changes in order to survive. These include responses to both environmental and physiological changes and autophagy is one of most important adaptive and regulatory mechanisms. Autophagy is defined as an autolytic process to clear damaged cellular organelles and recycle the nutrients via lysosomic degradation. The process of autophagy responds to special conditions such as nutrient withdrawal. Once autophagy is induced, phagophores form and then elongate and curve to form autophagosomes. Autophagosomes then engulf cargo, fuse with endosomes, and finally fuse with lysosomes for maturation. During the initiation process, the ATG1/ULK1 (unc-51-like kinase 1 and VPS34 (which encodes a class III phosphatidylinositol (PtdIns 3-kinase complexes are critical in recruitment and assembly of other complexes required for autophagy. The process of autophagy is regulated by autophagy related genes (ATGs. Amino acid and energy starvation mediate autophagy by activating mTORC1 (mammalian target of rapamycin and AMP-activated protein kinase (AMPK. AMPK is the energy status sensor, the core nutrient signaling component and the metabolic kinase of cells. This review mainly focuses on the mechanism of autophagy regulated by nutrient signaling especially for the two important complexes, ULK1 and VPS34.

  8. SIRT6 reduces macrophage foam cell formation by inducing autophagy and cholesterol efflux under ox-LDL condition.

    Science.gov (United States)

    He, Jiangping; Zhang, Guangya; Pang, Qi; Yu, Cong; Xiong, Jie; Zhu, Jing; Chen, Fengling

    2017-03-09

    SIRT6 is a pivotal regulator of lipid metabolism. It is also closely connected to cardiovascular diseases, which are the main cause of death in diabetic patients. We observed a decrease in the expression of SIRT6 and key autophagy effectors (ATG5, LC3B, and LAMP1) in ox-LDL-induced foam cells, a special form of lipid-laden macrophages. In these cells, SIRT6 WT but not SIRT6 H133Y overexpression markedly reduced foam cell formation, as shown by Oil Red O staining, while inducing autophagy flux, as determined by both mRFP-GFP-LC3 labeling and transmission electron microscopy. Silencing the key autophagy initiation gene ATG5, reversed the autophagy-promoting effect of SIRT6 in ox-LDL-treated THP1 cells, as evidenced by an increase in foam cells. Cholesterol efflux assays indicated that SIRT6 overexpression in foam cells promoted cholesterol efflux, increased the levels of ABCA1 and ABCG1, and reduced miR-33 levels. By transfecting miR-33 into cells overexpressing SIRT6, we observed that reduced foam cell formation and autophagy flux induction were largely reversed. These data imply that SIRT6 plays an essential role in protecting against atherosclerosis by reducing foam cell formation through an autophagy-dependent pathway. This article is protected by copyright. All rights reserved.

  9. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  10. 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.

  11. Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Noh, Mi Young; Kim, Dong Hyun; Kim, Iksoo; Han, Yeon Soo; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung

    2015-01-01

    Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model.

  12. MicroRNA-146a Induced by Hypoxia Promotes Chondrocyte Autophagy through Bcl-2

    Directory of Open Access Journals (Sweden)

    Fei Zhang

    2015-10-01

    Full Text Available Background/Aims: There have been many studies on the etiology of osteoarthritis (OA with regard to the function of inflammatory cytokines, the process of cartilage degradation, the function of miR-146a, hypoxia stimulation and autophagy in OA chondrocytes, but there have been no reports on the relationship between miR-146a and autophagy in cartilage, especially under hypoxia. This study aimed to confirm the relationship of miR-146a and autophagy in cartilage under hypoxia. Methods: Chondrocytes were treated by hypoxia gradients, and the main factors including HIF-1α, HIF-2α, miR-146a and Bcl-2 and autophagy markers ULK-1, ATG-5 were detected by quantitative PCR (Q-PCR and western blotting. The autophagy marker LC-3 was detected by immunofluorescence. The reciprocal effects between miR-146a and Bcl-2 were confirmed by several combinations of shRNAs and adenovirus-gene systems followed by Q-PCR and western blot detection. Results: Hypoxia maintained the chondrocytes phenotype and promoted autophagy and miR-146a expression via HIF-1α, but not HIF-2α, while miR-146a did not reversely affect HIF-1α. The autophagy induced by hypoxia through HIF-1α, miR-146a and Bcl-2. Simply, hypoxia induced HIF-1α, and HIF-1α increased miR-146a, but miR-146a suppressed Bcl-2, an autophagy inhibitor. While Bcl-2 affected neither HIF-1α nor miR-146a. The absence of both HIF-1α and miR-146a or Bcl-2 over-expression inhibited hypoxia-induced autophagy. Conclusion: HIF-1α, miR-146a and Bcl-2 play crucial roles during hypoxia-induced autophagy, Hypoxia, HIF-1α and miR-146a promote chondrocytes autophagy via depressing Bcl-2. We conclude that miR-146a may serve as a novel therapeutic target for protecting cartilage from degeneration in OA.

  13. 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.

  14. A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway.

    Directory of Open Access Journals (Sweden)

    Andrew M Arsham

    Full Text Available The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61alpha, and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1. We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms.

  15. Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.

    Science.gov (United States)

    Yang, Yue-Hua; Chen, Ke; Li, Bo; Chen, Jiang-Wei; Zheng, Xin-Feng; Wang, Yu-Ren; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2013-11-01

    Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through

  16. Ketogenic diet change cPLA2/clusterin and autophagy related gene expression and correlate with cognitive deficits and hippocampal MFs sprouting following neonatal seizures.

    Science.gov (United States)

    Ni, Hong; Zhao, Dong-Jing; Tian, Tian

    2016-02-01

    Because the ketogenic diet (KD) was affecting expression of energy metabolism- related genes in hippocampus and because lipid membrane peroxidation and its associated autophagy stress were also found to be involved in energy depletion, we hypothesized that KD might exert its neuroprotective action via lipid membrane peroxidation and autophagic signaling. Here, we tested this hypothesis by examining the long-term expression of lipid membrane peroxidation-related cPLA2 and clusterin, its downstream autophagy marker Beclin-1, LC3 and p62, as well as its execution molecule Cathepsin-E following neonatal seizures and chronic KD treatment. On postnatal day 9 (P9), 48 Sprague-Dawley rats were randomly assigned to two groups: flurothyl-induced recurrent seizures group and control group. On P28, they were further randomly divided into the seizure group without ketogenic diet (RS+ND), seizure plus ketogenic diet (RS+KD), the control group without ketogenic diet (NS+ND), and the control plus ketogenic diet (NS+KD). Morris water maze test was performed during P37-P43. Then mossy fiber sprouting and the protein levels were detected by Timm staining and Western blot analysis, respectively. Flurothyl-induced RS+ND rats show a long-term lower amount of cPLA2 and LC3II/I, and higher amount of clusterin, Beclin-1, p62 and Cathepsin-E which are in parallel with hippocampal mossy fiber sprouting and cognitive deficits. Furthermore, chronic KD treatment (RS+KD) is effective in restoring these molecular, neuropathological and cognitive changes. The results imply that a lipid membrane peroxidation and autophagy-associated pathway is involved in the aberrant hippocampal mossy fiber sprouting and cognitive deficits following neonatal seizures, which might be a potential target of KD for the treatment of neonatal seizure-induced brain damage.

  17. HIV-1 differentially modulates autophagy in neurons and astrocytes.

    Science.gov (United States)

    Mehla, Rajeev; Chauhan, Ashok

    2015-08-15

    Autophagy, a lysosomal degradative pathway that maintains cellular homeostasis, has emerged as an innate immune defense against pathogens. The role of autophagy in the deregulated HIV-infected central nervous system (CNS) is unclear. We have found that HIV-1-induced neuro-glial (neurons and astrocytes) damage involves modulation of the autophagy pathway. Neuro-glial stress induced by HIV-1 led to biochemical and morphological dysfunctions. X4 HIV-1 produced neuro-glial toxicity coupled with suppression of autophagy, while R5 HIV-1-induced toxicity was restricted to neurons. Rapamycin, a specific mTOR inhibitor (autophagy inducer) relieved the blockage of the autophagy pathway caused by HIV-1 and resulted in neuro-glial protection. Further understanding of the regulation of autophagy by cytokines and chemokines or other signaling events may lead to recognition of therapeutic targets for neurodegenerative diseases.

  18. 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.

  19. Tumor suppression and promotion by autophagy.

    Science.gov (United States)

    Ávalos, Yenniffer; Canales, Jimena; Bravo-Sagua, Roberto; Criollo, Alfredo; Lavandero, Sergio; Quest, Andrew F G

    2014-01-01

    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.

  20. Protective effects of astaxanthin on ConA-induced autoimmune hepatitis by the JNK/p-JNK pathway-mediated inhibition of autophagy and apoptosis.

    Directory of Open Access Journals (Sweden)

    Jingjing Li

    Full Text Available Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg, and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h. Primary hepatocytes were pretreated with astaxanthin (80 μM in vitro 24 h before stimulation with TNF-α (10 ng/ml. The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.

  1. Inhibition of Autophagy via Activation of PI3K/Akt Pathway Contributes to the Protection of Ginsenoside Rb1 against Neuronal Death Caused by Ischemic Insults

    Directory of Open Access Journals (Sweden)

    Tianfei Luo

    2014-09-01

    Full Text Available Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1 on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO and Monodansylcadaverine (MDC staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.

  2. Autophagy in lung disease pathogenesis and therapeutics

    Directory of Open Access Journals (Sweden)

    Stefan W. Ryter

    2015-04-01

    Full Text Available 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, and immune system functions. Selective pathways for autophagic degradation of cargoes may have variable significance in disease pathogenesis. Among these, the autophagic clearance of bacteria (xenophagy may represent a crucial host defense mechanism in the pathogenesis of sepsis and inflammatory diseases. Our recent studies indicate that the autophagic clearance of mitochondria, a potentially protective program, may aggravate the pathogenesis of chronic obstructive pulmonary disease by activating cell death programs. We report similar findings with respect to the autophagic clearance of cilia components, which can contribute to airways dysfunction in chronic lung disease. In certain diseases such as pulmonary hypertension, autophagy may confer protection by modulating proliferation and cell death. In other disorders, such as idiopathic pulmonary fibrosis and cystic fibrosis, impaired autophagy may contribute to pathogenesis. In lung cancer, autophagy has multiple consequences by limiting carcinogenesis, modulating therapeutic effectiveness, and promoting tumor cell survival. In this review we highlight the multiple functions of autophagy and its selective autophagy subtypes that may be of significance to the pathogenesis of human disease, with an emphasis on lung disease and therapeutics.

  3. Short-term starvation attenuates liver ischemia-reperfusion injury (IRI) by Sirt1-autophagy signaling in mice

    Science.gov (United States)

    Qin, Jianjie; Zhou, Junjin; Dai, Xinzheng; Zhou, Haoming; Pan, Xiongxiong; Wang, Xuehao; Zhang, Feng; Rao, Jianhua; Lu, Ling

    2016-01-01

    Calorie restriction or starvation (fasting) has some beneficial effects in terms of prolonging life and increasing resistance to stress. It has also been shown that calorie restriction has a protective role during ischemia-reperfusion injury (IRI) in several organs, but the underlying mechanism has not been elucidated. In this study we investigated the effects and molecular mechanisms of short-term starvation (STS) on liver IRI in a mouse liver IRI model. We found that STS significantly attenuated liver IRI in this model, as evidenced by inhibition of serum aminotransferase levels, and decreased pathological damage and hepatocellular apoptosis, especially after 2- or 3-day starvation. Furthermore, we found that 2- or 3-day starvation induced expression of hepatocellular autophagy in vivo and in vitro. Further experiments provided support for the notion that STS-induced autophagy played a key role during starvation-regulated protection against liver IRI via autophagy inhibition with 3-methyladenine. Interestingly, the longevity gene Sirt1 was also significantly up-regulated in liver after STS. Importantly, inhibition of Sirt1 by sirtinol abolished STS-induced autophagy and further abrogated STS-mediated protection against liver IRI. In conclusion, our results indicate that STS attenuates liver IRI via the Sirt1-autophagy pathway. Our findings provide a rationale for a novel therapeutic strategy for managing liver IRI. PMID:27648127

  4. Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells.

    Science.gov (United States)

    Wei, Ming-Feng; Chen, Min-Wei; Chen, Ke-Cheng; Lou, Pei-Jen; Lin, Susan Yun-Fan; Hung, Shih-Chieh; Hsiao, Michael; Yao, Cheng-Jung; Shieh, Ming-Jium

    2014-07-01

    Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133(+) cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133(+) cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.

  5. 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.

  6. Aggresome–Autophagy Involvement in a Sarcopenic Patient with Rigid Spine Syndrome and a p.C150R Mutation in FHL1 Gene

    Science.gov (United States)

    Sabatelli, Patrizia; Castagnaro, Silvia; Tagliavini, Francesca; Chrisam, Martina; Sardone, Francesca; Demay, Laurence; Richard, Pascale; Santi, Spartaco; Maraldi, Nadir M.; Merlini, Luciano; Sandri, Marco; Bonaldo, Paolo

    2014-01-01

    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, rigid spine syndrome (RSS), 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 RSS, and their mother had 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 multiprotein 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 in 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 involvement of the aggresome–autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation. PMID:25191266

  7. Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy

    Science.gov (United States)

    Wang, Jue; Cao, Bin; Han, Dong; Sun, Miao; Feng, Juan

    2017-01-01

    Long non-coding RNA H19 (lncRNA H19) was found to be upregulated by hypoxia, its expression and function have never been tested in cerebral ischemia and reperfusion (I/R) injury. This study intended to investigate the role of lncRNA H19 and H19 gene variation in cerebral I/R injury with focusing on its relationship with autophagy activation. Cerebral I/R was induced in rats by middle cerebral artery occlusion followed by reperfusion. SH-SY5Y cells were subjected to oxygen and glucose deprivation and reperfusion (OGD/R) to simulate I/R injury. Real-time PCR, flow cytometry, immunofluorescence and Western blot were used to evaluate the level of lncRNA H19, apoptosis, autophagy and some related proteins. The modified multiple ligase reaction was used to analyze the gene polymorphism of six SNPs in H19, rs217727, rs2067051, rs2251375, rs492994, rs2839698 and rs10732516 in ischemic stroke patients. We found that the expression of lncRNA H19 was upregulated by cerebral I/R in rats, as well as by OGD/R in vitro in the cells. Inhibition of lncRNA H19 and autophagy protected cells from OGD/R-induced death, respectively. Autophagy activation induced by OGD/R was prevented by H19 siRNA. Autophagy inducer, rapamycin, abolished lncRNA H19 effect. Furthermore, we found that lncRNA H19 inhibited autophagy through DUSP5-ERK1/2 axis. The result from blood samples of ischemic patients revealed that the variation of H19 gene increased the risk of ischemic stroke. Taken together, the results of present study suggest that LncRNA H19 could be a new therapeutic target of ischemic stroke. PMID:28203482

  8. Autophagy is an inflammation-related defensive mechanism against disease.

    Science.gov (United States)

    Joven, Jorge; Guirro, Maria; Mariné-Casadó, Roger; Rodríguez-Gallego, Esther; Menéndez, Javier A

    2014-01-01

    The inflammatory response is an energy-intensive process. Consequently, metabolism is closely associated with immune function. The autophagy machinery plays a role in metabolism by providing energy but may also be used to attack invading pathogens (xenophagy). The autophagy machinery may function to protect against not only the threats of infection but also the threats of the host's own response acting on the central immunological tolerance and the negative regulation of innate and inflammatory signaling. The balance between too little and too much autophagy is critical for the survival of immune cells because autophagy is linked to type 2-cell death programmed necrosis and apoptosis. Changes in inflammatory cells are driven by extracellular signals; however, the mechanisms by which cytokines mediate autophagy regulation and govern immune cell function remain unknown. Certain cytokines increase autophagy, whereas others inhibit autophagy. The relationship between autophagy and inflammation is also important in the pathogenesis of metabolic, non-communicable diseases. Inflammation per se is not the cause of obesity-associated diseases, but it is secondary to both the positive energy balance and the specific cellular responses. In metabolic tissues, the suppression of autophagy increases inflammation with the overexpression of cytokines, resulting in an activation of autophagy. The physiological role of these apparently contradictory findings remains uncertain but exemplifies future challenges in the therapeutic modulation of autophagy in the management of disease.

  9. Autophagy-related prognostic signature for breast cancer.

    Science.gov (United States)

    Gu, Yunyan; Li, Pengfei; Peng, Fuduan; Zhang, Mengmeng; Zhang, Yuanyuan; Liang, Haihai; Zhao, Wenyuan; Qi, Lishuang; Wang, Hongwei; Wang, Chenguang; Guo, Zheng

    2016-03-01

    Autophagy is a process that degrades intracellular constituents, such as long-lived or damaged proteins and organelles, to buffer metabolic stress under starvation conditions. Deregulation of autophagy is involved in the progression of cancer. However, the predictive value of autophagy for breast cancer prognosis remains unclear. First, based on gene expression profiling, we found that autophagy genes were implicated in breast cancer. Then, using the Cox proportional hazard regression model, we detected autophagy prognostic signature for breast cancer in a training dataset. We identified a set of eight autophagy genes (BCL2, BIRC5, EIF4EBP1, ERO1L, FOS, GAPDH, ITPR1 and VEGFA) that were significantly associated with overall survival in breast cancer. The eight autophagy genes were assigned as a autophagy-related prognostic signature for breast cancer. Based on the autophagy-related signature, the training dataset GSE21653 could be classified into high-risk and low-risk subgroups with significantly different survival times (HR = 2.72, 95% CI = (1.91, 3.87); P = 1.37 × 10(-5)). Inactivation of autophagy was associated with shortened survival of breast cancer patients. The prognostic value of the autophagy-related signature was confirmed in the testing dataset GSE3494 (HR = 2.12, 95% CI = (1.48, 3.03); P = 1.65 × 10(-3)) and GSE7390 (HR = 1.76, 95% CI = (1.22, 2.54); P = 9.95 × 10(-4)). Further analysis revealed that the prognostic value of the autophagy signature was independent of known clinical prognostic factors, including age, tumor size, grade, estrogen receptor status, progesterone receptor status, ERBB2 status, lymph node status and TP53 mutation status. Finally, we demonstrated that the autophagy signature could also predict distant metastasis-free survival for breast cancer.

  10. Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

    Science.gov (United States)

    Cabrera, Sandra; Maciel, Mariana; Herrera, Iliana; Nava, Teresa; Vergara, Fabián; Gaxiola, Miguel; López-Otín, Carlos; Selman, Moisés; Pardo, Annie

    2015-04-01

    Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

  11. 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

  12. Regulation of autophagy by the inositol trisphosphate receptor.

    Science.gov (United States)

    Criollo, A; Maiuri, M C; Tasdemir, E; Vitale, I; Fiebig, A A; Andrews, D; Molgó, J; Díaz, J; Lavandero, S; Harper, F; Pierron, G; di Stefano, D; Rizzuto, R; Szabadkai, G; Kroemer, G

    2007-05-01

    The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.

  13. Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

    Science.gov (United States)

    Sukseree, Supawadee; Chen, Ying-Ting; Laggner, Maria; Gruber, Florian; Petit, Valérie; Nagelreiter, Ionela-Mariana; Mlitz, Veronika; Rossiter, Heidemarie; Pollreisz, Andreas; Schmidt-Erfurth, Ursula; Larue, Lionel; Tschachler, Erwin

    2016-01-01

    Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms. PMID:27537685

  14. Autophagy, Metabolism, and Cancer.

    Science.gov (United States)

    White, Eileen; Mehnert, Janice M; Chan, Chang S

    2015-11-15

    Macroautophagy (autophagy hereafter) captures intracellular proteins and organelles and degrades them in lysosomes. The degradation breakdown products are released from lysosomes and recycled into metabolic and biosynthetic pathways. Basal autophagy provides protein and organelle quality control by eliminating damaged cellular components. Starvation-induced autophagy recycles intracellular components into metabolic pathways to sustain mitochondrial metabolic function and energy homeostasis. Recycling by autophagy is essential for yeast and mammals to survive starvation through intracellular nutrient scavenging. Autophagy suppresses degenerative diseases and has a context-dependent role in cancer. In some models, cancer initiation is suppressed by autophagy. By preventing the toxic accumulation of damaged protein and organelles, particularly mitochondria, autophagy limits oxidative stress, chronic tissue damage, and oncogenic signaling, which suppresses cancer initiation. This suggests a role for autophagy stimulation in cancer prevention, although the role of autophagy in the suppression of human cancer is unclear. In contrast, some cancers induce autophagy and are dependent on autophagy for survival. Much in the way that autophagy promotes survival in starvation, cancers can use autophagy-mediated recycling to maintain mitochondrial function and energy homeostasis to meet the elevated metabolic demand of growth and proliferation. Thus, autophagy inhibition may be beneficial for cancer therapy. Moreover, tumors are more autophagy-dependent than normal tissues, suggesting that there is a therapeutic window. Despite these insights, many important unanswered questions remain about the exact mechanisms of autophagy-mediated cancer suppression and promotion, how relevant these observations are to humans, and whether the autophagy pathway can be modulated therapeutically in cancer. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy."

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

    Science.gov (United States)

    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

    2011-02-21

    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 dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

  16. Autophagy: for better or for worse

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

    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.It is implicated in development,differentiation,innate and adaptive immunity,ageing and cell death.In addition,accumulating evidence demonstrates interesting links between autophagy and several human diseases and tumor development.Therefore,autophagy seems to be an important player in the life and death of cells and organisms.Despite the mounting knowledge about autophagy,the mechanisms through which the autophagic machinery regulates these diverse processes are not entirely understood.In this review,we give a comprehensive overview of the autophagic signaling pathway,its role in general cellular processes and its connection to cell death.In addition,we present a brief overview of the possible contribution of defective autophagic signaling to disease.

  17. 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.

  18. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

    Science.gov (United States)

    Wong, Vincent Kam Wai; Wu, An Guo; Wang, Jing Rong; Liu, Liang; Law, Betty Yuen-Kwan

    2015-02-18

    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.

  19. Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis.

    Science.gov (United States)

    Li, Wei-wei; Chen, Ming; Zhong, Li; Liu, Jia-ming; Xu, Zhao-shi; Li, Lian-cheng; Zhou, Yong-Bin; Guo, Chang-Hong; Ma, You-Zhi

    2015-12-25

    Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation.

  20. Autophagy in granular corneal dystrophy type 2.

    Science.gov (United States)

    Choi, Seung-Il; Kim, Eung Kweon

    2016-03-01

    Autophagy is a lysosomal degradative process that is essential for cellular homeostasis and metabolic stress adaptation. Defective autophagy is involved in the pathogenesis of many diseases including granular corneal dystrophy type 2 (GCD2). GCD2 is an autosomal dominant disorder caused by substitution of histidine for arginine at codon 124 (R124H) in the transforming growth factor β-induced gene (TGFBI) on chromosome 5q31. Transforming growth factor β-induced protein (TGFBIp) is degraded by autophagy, but mutant-TGFBIp accumulates in autophagosomes and/or lysosomes, despite significant activation of basal autophagy, in GCD2 corneal fibroblasts. Furthermore, inhibition of autophagy induces cell death of GCD2 corneal fibroblasts through active caspase-3. As there is currently no pharmacological treatment for GCD2, development of novel therapies is required. A potential strategy for preventing cytoplasmic accumulation of mutant-TGFBIp in GCD2 corneal fibroblasts is to enhance mutant-TGFBIp degradation. This could be achieved by activation of the autophagic pathway. Here, we will consider the role and the potential therapeutic benefits of autophagy in GCD2, with focus on TGFBIp degradation, in light of the recently established role of autophagy in protein degradation.

  1. Guidelines for monitoring autophagy in Caenorhabditis elegans.

    Science.gov (United States)

    Zhang, Hong; Chang, Jessica T; Guo, Bin; Hansen, Malene; Jia, Kailiang; Kovács, Attila L; Kumsta, Caroline; Lapierre, Louis R; Legouis, Renaud; Lin, Long; Lu, Qun; Meléndez, Alicia; O'Rourke, Eyleen J; Sato, Ken; Sato, Miyuki; Wang, Xiaochen; Wu, Fan

    2015-01-01

    The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

  2. E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy.

    Directory of Open Access Journals (Sweden)

    Madhavi Latha Somaraju Chalasani

    Full Text Available The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.

  3. Autophagy in Tuberculosis

    Science.gov (United States)

    Deretic, Vojo

    2014-01-01

    Autophagy as an immune mechanism controls inflammation and acts as a cell-autonomous defense against intracellular microbes including Mycobacterium tuberculosis. An equally significant role of autophagy is its anti-inflammatory and tissue-sparing function. This combination of antimicrobial and anti-inflammatory actions prevents active disease in animal models. In human populations, genetic links between autophagy, inflammatory bowel disease, and susceptibility to tuberculosis provide further support to these combined roles of autophagy. The autophagic control of M. tuberculosis and prevention of progressive disease provide novel insights into physiological and immune control of tuberculosis. It also offers host-based therapeutic opportunities because autophagy can be pharmacologically modulated. PMID:25167980

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

    Directory of Open Access Journals (Sweden)

    Silke Metzger

    Full Text Available 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, 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 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 modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients. In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis.

  5. Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy.

    Science.gov (United States)

    Bernard, Amélie; Klionsky, Daniel J

    2015-04-01

    To maintain proper cellular homeostasis, the magnitude of autophagy activity has to be finely tuned in response to environmental changes. Many aspects of autophagy regulation have been extensively studied: pathways integrating signals through the master regulators TORC1 and PKA lead to multiple post-translational modifications affecting the functions, protein-protein interactions, and localization of Atg proteins. The expression of several ATG genes increases sharply upon autophagy induction conditions, and defects in ATG gene expression are associated with various diseases, pointing to the importance of transcriptional regulation of autophagy. Yet, how changes in ATG gene expression affect the rate of autophagy is not well characterized, and transcriptional regulators of the autophagy pathway remain largely unknown. To identify such regulators, we analyzed the expression of several ATG genes in a library of DNA-binding protein mutants. This led to the identification of Rph1 as a master transcriptional regulator of autophagy.

  6. Cloning of the Protective Antigen Gene of Bacillus anthracis

    Science.gov (United States)

    1983-09-01

    of the complicated precedents of duplicate toxin genes in chro- muumm mosomall and plasmid DNA of B. thuringiensis (Schnepf and Whitely, 1981; Klier...OiL V4. 34. S-W7. SW 1v 99 CwI 0193 by MT 0 009-7483/06O-002.00/0 mU"- - 1*;)-0Cloning of the Protective Antigen Gene OCT 19 MI L Sof Bacillus ...Sumnler uncertain, it is probably caused by other Bacillus antigens, 4 t which may include LF and EF. PA produced from recom- A The - "w t of a

  7. 保护性自噬对5-FU诱导的胃癌细胞凋亡的抑制作用%Protective autophagy inhibits 5-FU-induced apoptosis in human gastric cancer cell line MGC803

    Institute of Scientific and Technical Information of China (English)

    徐玲; 曲秀娟; 刘云鹏; 刘静; 丁小娣; 侯科佐; 张晔

    2011-01-01

    目的:明确5-FU处理胃癌MGC803细胞后自噬的存在,并探讨自噬在5-FU诱导凋亡中的作用.方法:5-FU处理胃癌MGC803细胞.MTT检测细胞增殖能力.流式细胞术检测细胞凋亡.Western blot检测蛋白表达.荧光显微镜观察自噬的发生.结果:0.1-1000mg/L的5-FU作用MGC803细胞48h,抑制细胞增殖50%的药物浓度(IC50)为2.07mg/L±1.14mg/L.2mg/L和5mg/L的5-FU作用细胞48h,细胞凋亡率分别为22.46%±3.21%和32.27%±4.52%.同时,5-FU诱导细胞自噬的发生,观察到LC3的点状聚集和LC3-Ⅱ蛋白的提高.并且,5-FU抑制了PI3K/Akt/mTOR通路的活性.用氯喹抑制自噬明显提高了5-FU诱导的细胞调亡(P<0.05).结论:5-FU诱导保护性自噬并阻止了胃癌MGC803细胞的凋亡.5-FU和自噬抑制剂的联合应用可能是很有希望的胃癌治疗策略.%AIM: To investigate whether 5-fluorouracil (5-FU)induces autophagy in human gastric cancer cell line MGC803 and to identify the role of autophagy in 5-FU -induced cell apoptosis.METHODS: After cultured MGC803 ceils were treated with 5-FU, cell proliferation was measured using MTT assay; cell apoptosis was determined by flow cytometry; protein expression was detected by Western blot; and autophagy was observed by fluorescent microscopy.RESULTS: The concentration of 5-FU inducing a 50% inhibition of cell proliferation (IC50) was 2.07 mg/L ± 1.14 mg/L in MGC803 cells. After treatment with 2 and 5 mg/L 5-FU for 48 h, the rates of cell apoptosis were 22.46% ± 3.21% and 32.27% ± 4.52%, respectively. Autophagy, characterized by an increase in the number of punctate LC3 dots and the level of LC3-Ⅱ protein,was observed in cells treated with 5-FU. The activity of the PI3K/Akt/mTOR pathway was inhibited by 5-FU treatment. Inhibition of autophagy with chlorochine significantly enhanced 5-FU -induced apoptosis (P < 0.05).CONCLUSION: 5-FU-induced protective autophagy prevents MGC803 cells from apoptosis.Combination therapy with 5-FU and

  8. Autophagy controls BCG-induced trained immunity and the response to intravesical BCG therapy for bladder cancer.

    Directory of Open Access Journals (Sweden)

    Kathrin Buffen

    2014-10-01

    Full Text Available The anti-tuberculosis-vaccine Bacillus Calmette-Guérin (BCG is the most widely used vaccine in the world. In addition to its effects against tuberculosis, BCG vaccination also induces non-specific beneficial effects against certain forms of malignancy and against infections with unrelated pathogens. It has been recently proposed that the non-specific effects of BCG are mediated through epigenetic reprogramming of monocytes, a process called trained immunity. In the present study we demonstrate that autophagy contributes to trained immunity induced by BCG. Pharmacologic inhibition of autophagy blocked trained immunity induced in vitro by stimuli such as β-glucans or BCG. Single nucleotide polymorphisms (SNPs in the autophagy genes ATG2B (rs3759601 and ATG5 (rs2245214 influenced both the in vitro and in vivo training effect of BCG upon restimulation with unrelated bacterial or fungal stimuli. Furthermore, pharmacologic or genetic inhibition of autophagy blocked epigenetic reprogramming of monocytes at the level of H3K4 trimethylation. Finally, we demonstrate that rs3759601 in ATG2B correlates with progression and recurrence of bladder cancer after BCG intravesical instillation therapy. These findings identify a key role of autophagy for the nonspecific protective effects of BCG.

  9. Nanomaterials, Autophagy, and Lupus Disease.

    Science.gov (United States)

    Bianco, Alberto; Muller, Sylviane

    2016-01-19

    Nanoscale materials hold great promise in the therapeutic field. In particular, as carriers or vectors, they help bioactive molecules reach their primary targets. Furthermore, by themselves, certain nanomaterials-regarded as protective-can modulate particular metabolic pathways that are deregulated in pathological situations. They can also synergistically improve the effects of a payload drug. These properties are the basis of their appeal. However, nanoscale materials can also have intrinsic properties that limit their use, and this is the case for certain types of nanomaterials that influence autophagy. This property can be beneficial in some pathological settings, but in others, if the autophagic flux is already accelerated, it can be deleterious. This is notably the case for systemic lupus erythematosus (SLE) and other chronic inflammatory diseases, including certain neurological diseases. The nanomaterial-autophagy interaction therefore must be treated with caution for therapeutic molecules and peptides that require vectorization for their administration.

  10. RUFY4: Immunity piggybacking on autophagy?

    Science.gov (United States)

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

    2016-01-01

    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.

  11. 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; Dusek, Rachel L.; 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-...

  12. Autophagy is essential for cardiac morphogenesis during vertebrate development.

    Science.gov (United States)

    Lee, Eunmyong; Koo, Yeon; Ng, Aylwin; Wei, Yongjie; Luby-Phelps, Kate; Juraszek, Amy; Xavier, Ramnik J; Cleaver, Ondine; Levine, Beth; Amatruda, James F

    2014-04-01

    Genetic analyses indicate that autophagy, an evolutionarily conserved lysosomal degradation pathway, is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryogenesis. To address this question, we examined the role of autophagy in the early development of zebrafish, a model organism for studying vertebrate tissue and organ morphogenesis. Using zebrafish that transgenically express the fluorescent autophagy reporter protein, GFP-LC3, we found that autophagy is active in multiple tissues, including the heart, during the embryonic period. Inhibition of autophagy by morpholino knockdown of essential autophagy genes (including atg5, atg7, and becn1) resulted in defects in morphogenesis, increased numbers of dead cells, abnormal heart structure, and reduced organismal survival. Further analyses of cardiac development in autophagy-deficient zebrafish revealed defects in cardiac looping, abnormal chamber morphology, aberrant valve development, and ectopic expression of critical transcription factors including foxn4, tbx5, and tbx2. Consistent with these results, Atg5-deficient mice displayed abnormal Tbx2 expression and defects in valve development and chamber septation. Thus, autophagy plays an essential, conserved role in cardiac morphogenesis during vertebrate development.

  13. Application and interpretation of current autophagy inhibitors and activators

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Li-fang HU; Hui-fen ZHENG; Cheng-jie MAO; Wei-dong HU; Kang-ping XIONG; Fen WANG

    2013-01-01

    Aut ophagy is the major intracellular degradation system,by which cytoplasmic materials are delivered to and degraded in the lysosome.As a quality control mechanism for cytoplasmic proteins and organelles,autophagy plays important roles in a variety of human diseases,including neurodegenerative diseases,cancer,cardiovascular disease,diabetes and infectious and inflammatory diseases.The discovery of ATG genes and the dissection of the signaling pathways involved in regulating autophagy have greatly enriched our knowledge on the occurrence and development of this lysosomal degradation pathway.In addition to its role in degradation,autophagy may also promote a type of programmed cell death that is different from apoptosis,termed type II programmed cell death.Owing to the dual roles of autophagy in cell death and the specificity of diseases,the exact mechanisms of autophagy in various diseases require more investigation.The application of autophagy inhibitors and activators will help us understand the regulation of autophagy in human diseases,and provide insight into the use of autophagy-targeted drugs.In this review,we summarize the latest research on autophagy inhibitors and activators and discuss the possibility of their application in human disease therapy.

  14. 自噬在乙醇介导神经毒性中的保护作用及机制%The protective effects and mechanisms of autophagy on ethanol-mediated neurotoxicity

    Institute of Scientific and Technical Information of China (English)

    张硕(综述); 冯娟(校审)

    2015-01-01

    Ethanol exposure can result in neuronal death and neuronal degeneration, which are severely detrimental to the brain. hTe developing brain is particularly vulnerable to ethanol such that prenatal ethanol consumption causes fetal alcohol spectrum disorders;and long-term ethanol intake can lead to chronic alcoholic nervous disease in adults such as Wernicke-Korsakoff syndrome. One cellular mechanism that acts as a protective response is autophagy, which can alleviate ethanol-induced neuron apoptosis and other pathological responses. Ethanol may stimulate autophagy through multiple mechanisms including induction of oxidative stress and endoplasmic reticulum stress, modulation of MTOR and AMPK signaling. hTis review summarizes and discusses the recent progress and mechanisms regarding the involvement of autophagy in ethanol-mediated neurotoxicity.%乙醇暴露能够导致神经元变性及神经元死亡,引起严重的神经系统损伤。发育期的神经系统对乙醇更为敏感,产前摄入乙醇可导致胎儿酒精谱系障碍;在成人中,慢性乙醇摄入可导致Wernicke-Korsakoff综合征等慢性酒精中毒性神经疾病。自噬作为对乙醇毒性的保护性机制,它能够缓解乙醇介导的神经元凋亡及其他病理性反应。乙醇通过多种细胞分子机制激活自噬,包括介导氧化应激、内质网应激、mTOR及AMPK信号通路等。本文总结讨论目前自噬参与乙醇神经毒性相关机制及研究进展。

  15. 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.

  16. Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy.

    Science.gov (United States)

    Li, Jingjing; Chen, Kan; Li, Sainan; Feng, Jiao; Liu, Tong; Wang, Fan; Zhang, Rong; Xu, Shizan; Zhou, Yuqing; Zhou, Shunfeng; Xia, Yujing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2016-01-01

    Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis.

  17. Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

    Science.gov (United States)

    Ramundo, Silvia; Casero, David; Mühlhaus, Timo; Hemme, Dorothea; Sommer, Frederik; Crèvecoeur, Michèle; Rahire, Michèle; Schroda, Michael; Rusch, Jannette; Goodenough, Ursula; Pellegrini, Matteo; Perez-Perez, Maria Esther; Crespo, José Luis; Schaad, Olivier; Civic, Natacha; Rochaix, Jean David

    2014-05-01

    Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

  18. Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control[W

    Science.gov (United States)

    Ramundo, Silvia; Casero, David; Mühlhaus, Timo; Hemme, Dorothea; Sommer, Frederik; Crèvecoeur, Michèle; Rahire, Michèle; Schroda, Michael; Rusch, Jannette; Goodenough, Ursula; Pellegrini, Matteo; Perez-Perez, Maria Esther; Crespo, José Luis; Schaad, Olivier; Civic, Natacha; Rochaix, Jean David

    2014-01-01

    Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria. PMID:24879428

  19. 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.

  20. 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.

  1. A nonapoptotic role for CASP2/caspase 2: modulation of autophagy.

    Science.gov (United States)

    Tiwari, Meenakshi; Sharma, Lokendra K; Vanegas, Difernando; Callaway, Danielle A; Bai, Yidong; Lechleiter, James D; Herman, Brian

    2014-06-01

    CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2(-/-)) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2(-/-) cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer.

  2. The yeast chromatin remodeler Rsc1-RSC complex is required for transcriptional activation of autophagy-related genes and inhibition of the TORC1 pathway in response to nitrogen starvation.

    Science.gov (United States)

    Yu, Feifei; Imamura, Yuko; Ueno, Masaru; Suzuki, Sho W; Ohsumi, Yoshinori; Yukawa, Masashi; Tsuchiya, Eiko

    2015-09-01

    The yeast RSC, an ATP-dependent chromatin-remodeling complex, is essential for mitotic and meiotic growth. There are two distinct isoforms of this complex defined by the presence of either Rsc1 or Rsc2; however, the functional differences between these complexes are unclear. Here we show that the RSC complex containing Rsc1, but not Rsc2, functions in autophagy induction. Rsc1 was required not only for full expression of ATG8 mRNA but also for maintenance of Atg8 protein stability. Interestingly, decreased autophagic activity and Atg8 protein stability in rsc1Δ cells, but not the defect in ATG8 mRNA expression, were partially suppressed by deletion of TOR1. In addition, we found that rsc1Δ impaired the binding between the Rho GTPase Rho1 and the TORC1-specific component Kog1, which is required for down-regulation of TORC1 activity. These results suggest that the Rsc1-containing RSC complex plays dual roles in the proper induction of autophagy: 1) the transcriptional activation of autophagy-related genes independent of the TORC1 pathway and 2) the inactivation of TORC1, possibly through enhancement of Rho1-Kog1 binding.

  3. Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy

    Directory of Open Access Journals (Sweden)

    Li J

    2016-02-01

    Full Text Available Jingjing Li,1 Kan Chen,1 Sainan Li,1 Jiao Feng,1 Tong Liu,1 Fan Wang,1 Rong Zhang,1,2 Shizan Xu,1,2 Yuqing Zhou,1,3 Shunfeng Zhou,1,3 Yujing Xia,1 Jie Lu,1 Yingqun Zhou,1 Chuanyong Guo1 1Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 2The First Clinical Medical College of Nanjing Medical University, Nanjing, 3Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, People’s Republic of China Abstract: Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis. Keywords: liver cirrhosis, hepatic stellate cells, bile duct ligation

  4. 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.

  5. Role of autophagy in the pathogenesis of multiple sclerosis.

    Science.gov (United States)

    Liang, Peizhou; Le, Weidong

    2015-08-01

    Autophagy plays an important role in maintaining the cellular homeostasis. One of its functions is to degrade unnecessary organelles and proteins for energy recycling or amino-acids for cell survival. Ablation of autophagy leads to neurodegeneration. Multiple sclerosis (MS), a permanent neurological impairment typical of chronic inflammatory demyelinating disorder, is an auto-immune disease of the central nervous system (CNS). Autophagy is tightly linked to the innate and adaptive immune systems during the autoimmune process, and several studies have shown that autophagy directly participates in the progress of MS or experimental autoimmune encephalomyelitis (EAE, a mouse model of MS). Dysfunction of mitochondria that intensively influences the autophagy pathway is one of the important factors in the pathogenesis of MS. Autophagy-related gene (ATG) 5 and immune-related GTPase M (IRGM) 1 are increased, while ATG16L2 is decreased, in T-cells in EAE and active relapsing-remitting MS brains. Administration of rapamycin, an inhibitor of mammalian target of rapamycin ( mTOR), ameliorates relapsing-remitting EAE. Inflammation and oxidative stress are increased in MS lesions and EAE, but Lamp2 and the LC3-II/LC3-I ratio are decreased. Furthermore, autophagy in various glial cells plays important roles in regulating neuro-inflammation in the CNS, implying potential roles in MS. In this review, we discuss the role of autophagy in the peripheral immune system and the CNS in neuroinflammation associated with the pathogenesis of MS.

  6. Autophagy Is Associated with Pathogenesis of Haemophilus parasuis

    Science.gov (United States)

    Zhang, Yaning; Li, Yufeng; Yuan, Wentao; Xia, Yuting; Shen, Yijuan

    2016-01-01

    Haemophilus parasuis (H. parasuis) is a common commensal Gram-negative extracellular bacterium in the upper respiratory tract of swine, which can cause Glässer's disease in stress conditions. Research on the pathogenicity of H. parasuis has mainly focused on immune evasion and bacterial virulence factors, while few studies have examined the interactions of H. parasuis and its host. Autophagy is associated with the replication and proliferation of many pathogenic bacteria, but whether it plays a role during infection by H. parasuis is unknown. In this study, an adenovirus construct expressing GFP, RFP, and LC3 was used to infect H. parasuis. Western blotting, laser confocal microscopy, and electron microscopy showed that Hps5 infection induced obvious autophagy in PK-15 cells. In cells infected with strains of H. parasuis differing in invasiveness, the levels of autophagy were positively correlated with the presence of alive bacteria in PK-15 cells. In addition, autophagy inhibited the invasion of Hps5 in PK-15 cells. Autophagy related genes Beclin, Atg5 and Atg7 were silenced with RNA interference, the results showed that autophagy induced by H. parasuis infection is a classical pathway. Our observations demonstrate that H. parasuis can induce autophagy and that the levels of autophagy are associated with the presence of alive bacteria in cells, which opened novel avenues to further our understanding of H. parasuis-host interplay and pathogenesis. PMID:27703447

  7. Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

    Science.gov (United States)

    Cebollero, Eduardo; Gonzalez, Ramon

    2006-01-01

    Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed. PMID:16751523

  8. Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes.

    Science.gov (United States)

    Colasanti, T; Vomero, M; Alessandri, C; Barbati, C; Maselli, A; Camperio, C; Conti, F; Tinari, A; Carlo-Stella, C; Tuosto, L; Benincasa, D; Valesini, G; Malorni, W; Pierdominici, M; Ortona, E

    2014-05-29

    It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.

  9. Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy.

    Science.gov (United States)

    Chaudhary, Kapil; Shinde, Rahul; Liu, Haiyun; Gnana-Prakasam, Jaya P; Veeranan-Karmegam, Rajalakshmi; Huang, Lei; Ravishankar, Buvana; Bradley, Jillian; Kvirkvelia, Nino; McMenamin, Malgorzata; Xiao, Wei; Kleven, Daniel; Mellor, Andrew L; Madaio, Michael P; McGaha, Tracy L

    2015-06-15

    Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure. In this article, we show that Ab-mediated inflammatory kidney injury and renal disease in a mouse nephrotoxic serum nephritis model was inhibited by amino acid metabolism and a protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity with subsequent activation of a stress response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage recruitment to the kidney during the incipient stage of Ab-induced glomerular inflammation. Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted Ab-induced, self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1 activity or treating mice with a GCN2 agonist induced autophagy and protected mice from nephritic kidney damage. Finally, kidney tissue from patients with Ab-driven nephropathy showed increased IDO1 abundance and stress gene expression. Thus, these findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a critical negative feedback mechanism that limits inflammatory renal pathologic changes by inducing autophagy.

  10. Autophagy Is an Innate Mechanism Associated with Leprosy Polarization

    Science.gov (United States)

    Andrade, Priscila Ribeiro; Ferreira, Helen; Nery, José Augusto da Costa; Côrte-Real, Suzana; da Silva, Gilberto Marcelo Sperandio; Rosa, Patricia Sammarco; Fabri, Mario; Sarno, Euzenir Nunes

    2017-01-01

    Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages. PMID:28056107

  11. 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 chemical...... autophagy inhibitors on the specific productivity (qp), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine...... and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125...

  12. Autophagy and cytokines.

    Science.gov (United States)

    Harris, James

    2011-11-01

    Autophagy is a highly conserved homoeostatic mechanism for the lysosomal degradation of cytosolic constituents, including long-lived macromolecules, organelles and intracellular pathogens. Autophagosomes are formed in response to a number of environmental stimuli, including amino acid deprivation, but also by both host- and pathogen-derived molecules, including toll-like receptor ligands and cytokines. In particular, IFN-γ, TNF-α, IL-1, IL-2, IL-6 and TGF-β have been shown to induce autophagy, while IL-4, IL-10 and IL-13 are inhibitory. Moreover, autophagy can itself regulate the production and secretion of cytokines, including IL-1, IL-18, TNF-α, and Type I IFN. This review discusses the potentially pivotal roles of autophagy in the regulation of inflammation and the coordination of innate and adaptive immune responses.

  13. Autophagy in Trypanosomatids

    Directory of Open Access Journals (Sweden)

    Paul A. M. Michels

    2012-07-01

    Full Text Available Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds.

  14. Trehalose-mediated autophagy impairs the anti-viral function of human primary airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Qun Wu

    Full Text Available Human rhinovirus (HRV is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5 effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I and IFN-β promoter stimulator 1 (IPS-1, two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations.

  15. A critical role of autophagy in plant resistance to necrotrophic fungal pathogens.

    Science.gov (United States)

    Lai, Zhibing; Wang, Fei; Zheng, Zuyu; Fan, Baofang; Chen, Zhixiang

    2011-06-01

    Autophagy is a pathway for degradation of cytoplasmic components. In plants, autophagy plays an important role in nutrient recycling during nitrogen or carbon starvation, and in responses to abiotic stress. Autophagy also regulates age- and immunity-related programmed cell death, which is important in plant defense against biotrophic pathogens. Here we show that autophagy plays a critical role in plant resistance to necrotrophic pathogens. ATG18a, a critical autophagy protein in Arabidopsis, interacts with WRKY33, a transcription factor that is required for resistance to necrotrophic pathogens. Expression of autophagy genes and formation of autophagosomes are induced in Arabidopsis by the necrotrophic fungal pathogen Botrytis cinerea. Induction of ATG18a and autophagy by B. cinerea was compromised in the wrky33 mutant, which is highly susceptible to necrotrophic pathogens. Arabidopsis mutants defective in autophagy exhibit enhanced susceptibility to the necrotrophic fungal pathogens B. cinerea and Alternaria brassicicola based on increased pathogen growth in the mutants. The hypersusceptibility of the autophagy mutants was associated with reduced expression of the jasmonate-regulated PFD1.2 gene, accelerated development of senescence-like chlorotic symptoms, and increased protein degradation in infected plant tissues. These results strongly suggest that autophagy cooperates with jasmonate- and WRKY33-mediated signaling pathways in the regulation of plant defense responses to necrotrophic pathogens.

  16. Intestinal autophagy activity is essential for host defense against Salmonella typhimurium infection in Caenorhabditis elegans.

    Science.gov (United States)

    Curt, Alexander; Zhang, Jiuli; Minnerly, Justin; Jia, Kailiang

    2014-08-01

    Salmonella typhimurium infects both intestinal epithelial cells and macrophages. Autophagy is a lysosomal degradation pathway that is present in all eukaryotes. Autophagy has been reported to limit the Salmonella replication in Caenorhabditis elegans and in mammals. However, it is unknown whether intestinal autophagy activity plays a role in host defense against Salmonella infection in C. elegans. In this study, we inhibited the autophagy gene bec-1 in different C. elegans tissues and examined the survival of these animals following Salmonella infection. Here we show that inhibition of the bec-1 gene in the intestine but not in other tissues confers susceptibility to Salmonella infection, which is consistent with recent studies in mice showing that autophagy is involved in clearance of Salmonella in the intestinal epithelial cells. Therefore, the intestinal autophagy activity is essential for host defense against Salmonella infection from C. elegans to mice, perhaps also in humans.

  17. Autophagy research: Lessons from metabolism

    NARCIS (Netherlands)

    A.J. Meijer

    2009-01-01

    Autophagy research continues to expand exponentially. Clearly autophagy and metabolism are intimately connected; however, the rapid expansion of research into this topic inevitably brings the risk that important basic knowledge of metabolism will be overlooked when considering experimental data. Unf

  18. The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana.

    Science.gov (United States)

    Ying, Sheng-Hua; Liu, Jing; Chu, Xin-Ling; Xie, Xue-Qin; Feng, Ming-Guang

    2016-05-20

    Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence.

  19. A dual role of p53 in the control of autophagy.

    Science.gov (United States)

    Tasdemir, Ezgi; Chiara Maiuri, M; Morselli, Eugenia; Criollo, Alfredo; D'Amelio, Marcello; Djavaheri-Mergny, Mojgan; Cecconi, Francesco; Tavernarakis, Nektarios; Kroemer, Guido

    2008-08-01

    Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53(-/-) cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g., starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On the one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy.

  20. 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.

  1. Species-specific impact of the autophagy machinery on Chikungunya virus infection.

    Science.gov (United States)

    Judith, Delphine; Mostowy, Serge; Bourai, Mehdi; Gangneux, Nicolas; Lelek, Mickaël; Lucas-Hourani, Marianne; Cayet, Nadège; Jacob, Yves; Prévost, Marie-Christine; Pierre, Philippe; Tangy, Frédéric; Zimmer, Christophe; Vidalain, Pierre-Olivier; Couderc, Thérèse; Lecuit, Marc

    2013-06-01

    Chikungunya virus (CHIKV) is a recently re-emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52--but not its mouse orthologue--interacts with the non-structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.

  2. Autophagy in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Kadija; Abounit; Tiziano; M; Scarabelli; Roy; B; McCauley

    2012-01-01

    Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The preautophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5’-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an impor-tant cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.

  3. 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.

  4. 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.

  5. Trehalose Accumulation Triggers Autophagy during Plant Desiccation.

    Directory of Open Access Journals (Sweden)

    Brett Williams

    2015-12-01

    Full Text Available Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis, by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.

  6. Avermectin induced autophagy in pigeon spleen tissues.

    Science.gov (United States)

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

    2015-12-05

    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.

  7. Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53/TRP53 status.

    Science.gov (United States)

    Yang, Annan; Kimmelman, Alec C

    2014-09-01

    Basal levels of autophagy are elevated in most pancreatic ductal adenocarcinomas (PDAC). Suppressing autophagy pharmacologically using chloroquine (CQ) or genetically with RNAi to essential autophagy genes inhibits human pancreatic cancer growth in vitro and in vivo, which presents possible treatment opportunities for PDAC patients using the CQ-derivative hydroxychloroquine (HCQ). Indeed, such clinical trials are ongoing. However, autophagy is a complex cellular mechanism to maintain cell homeostasis under stress. Based on its biological role, a dual role of autophagy in tumorigenesis has been proposed: at tumor initiation, autophagy helps maintain genomic stability and prevent tumor initiation; while in advanced disease, autophagy degrades and recycles cellular components to meet the metabolic needs for rapid growth. This model was proven to be the case in mouse lung tumor models. However, in contrast to prior work in various PDAC model systems, loss of autophagy in PDAC mouse models with embryonic homozygous Trp53 deletion does not inhibit tumor growth and paradoxically increases progression. This raised concerns whether there may be a genotype-dependent reliance of PDAC on autophagy. In a recent study, our group used a Trp53 heterozygous mouse PDAC model and human PDX xenografts to address the question. Our results demonstrate that autophagy inhibition was effective against PDAC tumors irrespective of TP53/TRP53 status.

  8. Inflammasome-independent modulation of cytokine response by autophagy in human cells.

    Directory of Open Access Journals (Sweden)

    Tania O Crişan

    Full Text Available Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient mice have shown that disruption of autophagy increases processing of IL-1β and IL-18 through an inflammasome-dependent manner. Using complementary approaches either inducing or inhibiting autophagy, we describe modulatory effects of autophagy on proinflammatory cytokine production in human cells. Inhibition of basal autophagy in human peripheral blood mononuclear cells (PBMCs significantly enhances IL-1β after stimulation with TLR2 or TLR4 ligands, while at the same time reducing the production of TNFα. In line with this, induction of autophagy by starvation inhibited IL-1β production. These effects of autophagy were not exerted at the processing step, as inflammasome activation was not influenced. In contrast, the effect of autophagy on cytokine production was on transcription level, and possibly involving the inhibition of p38 mitogen activated protein kinase (MAPK phosphorylation. In conclusion, autophagy modulates the secretion of proinflammatory cytokines in human cells through an inflammasome-independent pathway, and this is a novel mechanism that may be targeted in inflammatory diseases.

  9. A pathway of targeted autophagy is induced by DNA damage in budding yeast

    Science.gov (United States)

    Eapen, Vinay V.; Waterman, David P.; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G.; Loewith, Robbie J.; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J.; Haber, James E.

    2017-01-01

    Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response. PMID:28154131

  10. 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.

  11. 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.

  12. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    Directory of Open Access Journals (Sweden)

    Ying Xu

    2016-04-01

    Full Text Available It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN. Here we used shRNA transfection to knockdown the insulin receptor (IR gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

  13. Evolutionary trends and functional anatomy of the human expanded autophagy network.

    Science.gov (United States)

    Till, Andreas; Saito, Rintaro; Merkurjev, Daria; Liu, Jing-Jing; Syed, Gulam Hussain; Kolnik, Martin; Siddiqui, Aleem; Glas, Martin; Scheffler, Björn; Ideker, Trey; Subramani, Suresh

    2015-01-01

    All eukaryotic cells utilize autophagy for protein and organelle turnover, thus assuring subcellular quality control, homeostasis, and survival. In order to address recent advances in identification of human autophagy associated genes, and to describe autophagy on a system-wide level, we established an autophagy-centered gene interaction network by merging various primary data sets and by retrieving respective interaction data. The resulting network ('AXAN') was analyzed with respect to subnetworks, e.g. the prime gene subnetwork (including the core machinery, signaling pathways and autophagy receptors) and the transcription subnetwork. To describe aspects of evolution within this network, we assessed the presence of protein orthologs across 99 eukaryotic model organisms. We visualized evolutionary trends for prime gene categories and evolutionary tracks for selected AXAN genes. This analysis confirms the eukaryotic origin of autophagy core genes while it points to a diverse evolutionary history of autophagy receptors. Next, we used module identification to describe the functional anatomy of the network at the level of pathway modules. In addition to obvious pathways (e.g., lysosomal degradation, insulin signaling) our data unveil the existence of context-related modules such as Rho GTPase signaling. Last, we used a tripartite, image-based RNAi - screen to test candidate genes predicted to play a role in regulation of autophagy. We verified the Rho GTPase, CDC42, as a novel regulator of autophagy-related signaling. This study emphasizes the applicability of system-wide approaches to gain novel insights into a complex biological process and to describe the human autophagy pathway at a hitherto unprecedented level of detail.

  14. [Regulation of autophagy on dendritic cells during rat liver regeneration by IPA].

    Science.gov (United States)

    Qiwen, Wang; Wei, Jin; Cuifang, Chang; Cunshuan, Xu

    2015-03-01

    To understand the mechanism underlying autophagy in regulating dendritic cells during rat liver regeneration, we used the method of percoll density gradient centrifugation combined with immunomagnetic bead to isolate dendritic cells, the Rat Genome 230 2.0 Array to determine the expression changes of autophagy-related genes, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the autophagy activities. The results indicated that LC3, BECN1, ATG7 and SQSTM1 genes had significant expression changes during rat liver regeneration. There were 593 genes related to autophagy, among which 210 genes were identified as significant. We also showed that the activity of autophagy was enhanced in the priming phase and teminal phase of liver regeneration, weakened in the proliferative stage by comparative analysis method of IPA. The autophagy-related physiological activities mainly included RNA expression, RNA transcription, cell differentiation and proliferation, involving in PPARα/RXRα activation, acute phase response signaling, TREM1 signaling, IL-6 signaling, IL-8 signaling and IL-1 signaling, whose activities were increased or decreased in liver regeneration. Cluster analysis found that P53 and AMPK signaling participated in the regulation of dendritic cells autophagy, with AMPK signaling in the priming phase of liver regeneration, and both signaling pathways in the terminal phase. We conclude that dendritic cells autophagy played an important role in initiation of the immune response in priming phase and depletion of dendritic cells in late phase during rat liver regeneration.

  15. Inflammasome-independent modulation of cytokine response by autophagy in human cells

    NARCIS (Netherlands)

    Crisan, T.O.; Plantinga, T.S.; Veerdonk, F.L. van de; Farcas, M.F.; Stoffels, M.; Kullberg, B.J.; Meer, J.W. van der; Joosten, L.A.B.; Netea, M.G.

    2011-01-01

    Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient m

  16. 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.

  17. 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.

  18. DNA damage and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

    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.

  19. A matter of balance between life and death: targeting reactive oxygen species (ROS)-induced autophagy for cancer therapy.

    Science.gov (United States)

    Gibson, Spencer B

    2010-10-01

    Reactive oxygen species (ROS) have been implicated in many biological functions and diseases. Often their role is counterintuitive, where ROS can either promote cell survival or cell death depending on the cellular context. Similarly, autophagy is involved in many biological functions and diseases where it can either promote cell survival or cell death. There is now a growing consensus that ROS controls autophagy in multiple contexts and cell types. Furthermore, alterations in ROS and autophagy regulation contribute to cancer initiation and progression. However, how ROS and autophagy contribute to cancer and how to target either for cancer treatment is controversial. Blocking ROS generation could prevent cancer initiation, whereas blockage of autophagy seems to be required for initiation of cancer. In cancer progression, high levels of ROS correspond with increased metabolism and under metabolic stress autophagy is required to maintain cellular integrity. In cancer treatment, therapeutic drugs that increase ROS and autophagy have been implicated in their mechanism for cell death, such as 2-methoxyestrodial (2-ME) and arsenic trioxide (As(2)O(3)), whereas other therapeutic drugs that induce ROS and autophagy seem to have a protective effect. This has led to different approaches to treat cancer patients where autophagy is either activated or inhibited. Both views of ROS and autophagy are valid and reflect the balance within a cell to either survive or die. Understanding this balancing act within a cell is essential to determine whether to block or activate ROS-controlled autophagy for cancer therapy.

  20. Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

    Institute of Scientific and Technical Information of China (English)

    Min XU; Hui-ling ZHANG

    2011-01-01

    Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.

  1. Crosstalk between apoptosis and autophagy within the Beclin 1 interactome.

    Science.gov (United States)

    Maiuri, Maria Chiara; Criollo, Alfredo; Kroemer, Guido

    2010-02-03

    Although the essential genes for autophagy (Atg) have been identified, the molecular mechanisms through which Atg proteins control 'self eating' in mammalian cells remain elusive. Beclin 1 (Bec1), the mammalian orthologue of yeast Atg6, is part of the class III phosphatidylinositol 3-kinase (PI3K) complex that induces autophagy. The first among an increasing number of Bec1-interacting proteins that has been identified is the anti-apoptotic protein Bcl-2. The dissociation of Bec1 from Bcl-2 is essential for its autophagic activity, and Bcl-2 only inhibits autophagy when it is present in the endoplasmic reticulum (ER). A paper in this issue of the EMBO Journal has identified a novel protein, NAF-1 (nutrient-deprivation autophagy factor-1), that binds Bcl-2 at the ER. NAF-1 is a component of the inositol-1,4,5 trisphosphate (IP3) receptor complex, which contributes to the interaction of Bcl-2 with Bec1 and is required for Bcl-2 to functionally antagonize Bec1-mediated autophagy. This work provides mechanistic insights into how autophagy- and apoptosis-regulatory molecules crosstalk at the ER.

  2. FNDC5 Alleviates Hepatosteatosis by Restoring AMPK/mTOR-Mediated Autophagy, Fatty Acid Oxidation, and Lipogenesis in Mice.

    Science.gov (United States)

    Liu, Tong-Yan; Xiong, Xiao-Qing; Ren, Xing-Sheng; Zhao, Ming-Xia; Shi, Chang-Xiang; Wang, Jue-Jin; Zhou, Ye-Bo; Zhang, Feng; Han, Ying; Gao, Xing-Ya; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-11-01

    Fibronectin type III domain-containing 5 (FNDC5) protein induces browning of subcutaneous fat and mediates the beneficial effects of exercise on metabolism. However, whether FNDC5 is associated with hepatic steatosis, autophagy, fatty acid oxidation (FAO), and lipogenesis remains unknown. Herein, we show the roles and mechanisms of FNDC5 in hepatic steatosis, autophagy, and lipid metabolism. Fasted FNDC5(-/-) mice exhibited severe steatosis, reduced autophagy, and FAO, and enhanced lipogenesis in the liver compared with wild-type mice. Energy deprivation-induced autophagy, FAO, and AMPK activity were attenuated in FNDC5(-/-) hepatocytes, which were restored by activating AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Inhibition of mammalian target of rapamycin (mTOR) complex 1 with rapamycin enhanced autophagy and FAO and attenuated lipogenesis and steatosis in FNDC5(-/-) livers. FNDC5 deficiency exacerbated hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. Exogenous FNDC5 stimulated autophagy and FAO gene expression in hepatocytes and repaired the attenuated autophagy and palmitate-induced steatosis in FNDC5(-/-) hepatocytes. FNDC5 overexpression prevented hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. These results indicate that FNDC5 deficiency impairs autophagy and FAO and enhances lipogenesis via the AMPK/mTOR pathway. FNDC5 deficiency aggravates whereas FNDC5 overexpression prevents the HFD-induced hyperlipemia, hepatic lipid accumulation, and impaired FAO and autophagy in the liver.

  3. Autophagy resolves early retinal inflammation in Igf1-deficient mice

    Directory of Open Access Journals (Sweden)

    Ana I. Arroba

    2016-09-01

    Full Text Available Insulin-like growth factor-1 (IGF-1 is a growth factor with differentiating, anti-apoptotic and metabolic functions in the periphery, and anti-inflammatory properties in the nervous system. Mice that have mutations in the Igf1 gene, rendering the gene product inactive (Igf1−/−, present with age-related visual loss accompanied by structural alterations in the first synapses of the retinal pathway. Recent advances have revealed a crucial role of autophagy in immunity and inflammation. Keeping in mind this close relationship, we aimed to decipher these processes in the context of the defects that occur during ageing in the retina of Igf1−/− mice. Tnfa and Il1b mRNAs, and phosphorylation of JNK and p38 MAPK were elevated in the retinas of 6- and 12-month old Igf1−/− mice compared to those in age-matched Igf1+/+ controls. In 6-month-old Igf1−/− retinas, increased mRNA levels of the autophagy mediators Becn1, Atg9, Atg5 and Atg4, decreased p62 (also known as SQSTM1 protein expression together with an increased LC3-II:LC3-I ratio reflected active autophagic flux. However, in retinas from 12-month-old Igf1−/− mice, Nlrp3 mRNA, processing of the IL1β pro-form and immunostaining of active caspase-1 were elevated compared to those in age-matched Igf1+/+ controls, suggesting activation of the inflammasome. This effect concurred with accumulation of autophagosomes and decreased autophagic flux in the retina. Microglia localization and status of activation in the retinas of 12-month-old Igf1+/+ and Igf1−/− mice, analyzed by immunostaining of Cd11b and Iba-1, showed a specific distribution pattern in the outer plexiform layer (OPL, inner plexiform layer (IPL and inner nuclear layer (INL, and revealed an increased number of activated microglia cells in the retina of 12-month-old blind Igf1−/− mice. Moreover, reactive gliosis was exclusively detected in the retinas from 12-month-old blind Igf1−/− mice. In conclusion, this study

  4. Autophagy resolves early retinal inflammation in Igf1-deficient mice.

    Science.gov (United States)

    Arroba, Ana I; Rodríguez-de la Rosa, Lourdes; Murillo-Cuesta, Silvia; Vaquero-Villanueva, Laura; Hurlé, Juan M; Varela-Nieto, Isabel; Valverde, Ángela M

    2016-09-01

    Insulin-like growth factor-1 (IGF-1) is a growth factor with differentiating, anti-apoptotic and metabolic functions in the periphery, and anti-inflammatory properties in the nervous system. Mice that have mutations in the Igf1 gene, rendering the gene product inactive (Igf1(-/-)), present with age-related visual loss accompanied by structural alterations in the first synapses of the retinal pathway. Recent advances have revealed a crucial role of autophagy in immunity and inflammation. Keeping in mind this close relationship, we aimed to decipher these processes in the context of the defects that occur during ageing in the retina of Igf1(-/-) mice. Tnfa and Il1b mRNAs, and phosphorylation of JNK and p38 MAPK were elevated in the retinas of 6- and 12-month old Igf1(-/-) mice compared to those in age-matched Igf1(+/+) controls. In 6-month-old Igf1(-/-) retinas, increased mRNA levels of the autophagy mediators Becn1, Atg9, Atg5 and Atg4, decreased p62 (also known as SQSTM1) protein expression together with an increased LC3-II:LC3-I ratio reflected active autophagic flux. However, in retinas from 12-month-old Igf1(-/-) mice, Nlrp3 mRNA, processing of the IL1β pro-form and immunostaining of active caspase-1 were elevated compared to those in age-matched Igf1(+/+) controls, suggesting activation of the inflammasome. This effect concurred with accumulation of autophagosomes and decreased autophagic flux in the retina. Microglia localization and status of activation in the retinas of 12-month-old Igf1(+/+) and Igf1(-/-) mice, analyzed by immunostaining of Cd11b and Iba-1, showed a specific distribution pattern in the outer plexiform layer (OPL), inner plexiform layer (IPL) and inner nuclear layer (INL), and revealed an increased number of activated microglia cells in the retina of 12-month-old blind Igf1(-/-) mice. Moreover, reactive gliosis was exclusively detected in the retinas from 12-month-old blind Igf1(-/-) mice. In conclusion, this study provides new evidence in

  5. Autophagy resolves early retinal inflammation in Igf1-deficient mice

    Science.gov (United States)

    Rodríguez-de la Rosa, Lourdes; Murillo-Cuesta, Silvia; Vaquero-Villanueva, Laura; Hurlé, Juan M.; Varela-Nieto, Isabel; Valverde, Ángela M.

    2016-01-01

    ABSTRACT Insulin-like growth factor-1 (IGF-1) is a growth factor with differentiating, anti-apoptotic and metabolic functions in the periphery, and anti-inflammatory properties in the nervous system. Mice that have mutations in the Igf1 gene, rendering the gene product inactive (Igf1−/−), present with age-related visual loss accompanied by structural alterations in the first synapses of the retinal pathway. Recent advances have revealed a crucial role of autophagy in immunity and inflammation. Keeping in mind this close relationship, we aimed to decipher these processes in the context of the defects that occur during ageing in the retina of Igf1−/− mice. Tnfa and Il1b mRNAs, and phosphorylation of JNK and p38 MAPK were elevated in the retinas of 6- and 12-month old Igf1−/− mice compared to those in age-matched Igf1+/+ controls. In 6-month-old Igf1−/− retinas, increased mRNA levels of the autophagy mediators Becn1, Atg9, Atg5 and Atg4, decreased p62 (also known as SQSTM1) protein expression together with an increased LC3-II:LC3-I ratio reflected active autophagic flux. However, in retinas from 12-month-old Igf1−/− mice, Nlrp3 mRNA, processing of the IL1β pro-form and immunostaining of active caspase-1 were elevated compared to those in age-matched Igf1+/+ controls, suggesting activation of the inflammasome. This effect concurred with accumulation of autophagosomes and decreased autophagic flux in the retina. Microglia localization and status of activation in the retinas of 12-month-old Igf1+/+ and Igf1−/− mice, analyzed by immunostaining of Cd11b and Iba-1, showed a specific distribution pattern in the outer plexiform layer (OPL), inner plexiform layer (IPL) and inner nuclear layer (INL), and revealed an increased number of activated microglia cells in the retina of 12-month-old blind Igf1−/− mice. Moreover, reactive gliosis was exclusively detected in the retinas from 12-month-old blind Igf1−/− mice. In conclusion, this study

  6. Foxk proteins repress the initiation of starvation-induced atrophy and autophagy programs.

    Science.gov (United States)

    Bowman, Christopher John; Ayer, Donald E; Dynlacht, Brian David

    2014-12-01

    Autophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A-HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes through Foxk-Sin3-mediated transcriptional control.

  7. Nutritional Status and Cardiac Autophagy

    Directory of Open Access Journals (Sweden)

    Jihyun Ahn

    2013-02-01

    Full Text Available Autophagy is necessary for the degradation of long-lasting proteins and nonfunctional organelles, and is activated to promote cellular survival. However, overactivation of autophagy may deplete essential molecules and organelles responsible for cellular survival. Lifelong calorie restriction by 40% has been shown to increase the cardiac expression of autophagic markers, which suggests that it may have a cardioprotective effect by decreasing oxidative damage brought on by aging and cardiovascular diseases. Although cardiac autophagy is critical to regulating protein quality and maintaining cellular function and survival, increased or excessive autophagy may have deleterious effects on the heart under some circumstances, including pressure overload-induced heart failure. The importance of autophagy has been shown in nutrient supply and preservation of energy in times of limitation, such as ischemia. Some studies have suggested that a transition from obesity to metabolic syndrome may involve progressive changes in myocardial inflammation, mitochondrial dysfunction, fibrosis, apoptosis, and myocardial autophagy.

  8. 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.

  9. 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.

  10. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization.

    Science.gov (United States)

    Liu, Kun; Zhao, Enpeng; Ilyas, Ghulam; Lalazar, Gadi; Lin, Yu; Haseeb, Muhammad; Tanaka, Kathryn E; Czaja, Mark J

    2015-01-01

    Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

  11. MicroRNA regulation of Autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lund, Anders H

    2012-01-01

    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 small non......-coding RNAs have been closely linked to virtually all known fundamental biological pathways. Deregulation of miRNAs can contribute to the development of human diseases, including cancer, where they can function as bona fide oncogenes or tumor suppressors.In this review, we highlight recent advances linking miRNAs...... perspective, but also from a therapeutic view, where miRNAs can be harnessed experimentally to alter autophagy levels in human tumors, affecting parameters such as tumor survival and treatment sensitivity....

  12. Blockage of Autophagy in C6 Glioma Cells Enhanced Radiosensitivity Possibly by Attenuating DNA-PK-Dependent DSB Due to Limited Ku Nuclear Translocation and DNA Binding.

    Science.gov (United States)

    Liu, C; He, W; Jin, M; Li, H; Xu, H; Liu, H; Yang, K; Zhang, T; Wu, G; Ren, J

    2015-01-01

    Glioblastoma multiforme (GBM) is the most lethal brain tumor and notorious for its resistance to ionizing radiation (IR). Recent evidence suggests that one possible mechanism that enables resistance to IR and protects cells against therapeutic stress is cellular autophagy. The molecular basis for this pro-survival function, however, remains elusive. Herein, we report a molecular mechanism by which IR-induced autophagy accelerates the repair of DNA double-strand breaks (DSB). We demonstrate that IR induces the accumulation of autophagosomes, which is accompanied by elevated expression of autophagyrelated genes beclin-1, atg5, atg7, and atg12. Beclin-1 knockdown impaired the induction of IR-mediated autophagy and significantly sensitized glioma cells to radiation therapy in vitro and in vivo. Furthermore, our data is the first to demonstrate that the radiosensitizing effect of beclin-1 knockdown may result from the disruption of nuclear translocation and DNA binding activity of Ku proteins and consequent attenuation of DSB repair. Our findings help advance our understanding of the molecular mechanisms underlying IR-induced autophagy and provide a promising adjunctive therapeutic strategy for the radiosensitization of malignant glioma.

  13. 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

    2015-12-05

    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.

  14. Dysregulated autophagy increased melanocyte sensitivity to H2O2-induced oxidative stress in vitiligo

    Science.gov (United States)

    He, Yuanmin; Li, Shuli; Zhang, Weigang; Dai, Wei; Cui, Tingting; Wang, Gang; Gao, Tianwen; Li, Chunying

    2017-01-01

    In vitiligo, melanocytes are particularly vulnerable to oxidative stress owing to the pro-oxidant state generated during melanin synthesis and to the genetic antioxidant defects. Autophagy is a controlled self-digestion process which can protect cells against oxidative damage. However, the exact role of autophagy in vitiligo melanocytes in response to oxidative stress and the mechanism involved are still not clear. To determine the implications of autophagy for melanocyte survival in response to oxidative stress, we first detected the autophagic flux in normal melanocytes exposure to H2O2, and found that autophagy was significantly enhanced in normal melanocytes, for protecting cells against H2O2-induced oxidative damage. Nevertheless, vitiligo melanocytes exhibited dysregulated autophagy and hypersensitivity to H2O2-induced oxidative injury. In addition, we confirmed that the impairment of Nrf2-p62 pathway is responsible for the defects of autophagy in vitiligo melanocytes. Noteworthily, upregulation of the Nrf2-p62 pathway or p62 reduced H2O2-induced oxidative damage of vitiligo melanocytes. Therefore, our data demonstrated that dysregulated autophagy owing to the impairment of Nrf2-p62 pathway increase the sensitivity of vitiligo melanocytes to oxidative stress, thus promote the development of vitiligo. Upregulation of p62-dependent autophagy may be applied to vitiligo treatment in the future. PMID:28186139

  15. Targeting autophagy in neurodegenerative diseases.

    Science.gov (United States)

    Vidal, René L; Matus, Soledad; Bargsted, Leslie; Hetz, Claudio

    2014-11-01

    The most prevalent neurodegenerative disorders involve protein misfolding and the aggregation of specific proteins. Autophagy is becoming an attractive target to treat neurodegenerative disorders through the selective degradation of abnormally folded proteins by the lysosomal pathway. However, accumulating evidence indicates that autophagy impairment at different regulatory steps may contribute to the neurodegenerative process. Thus, a complex scenario is emerging where autophagy may play a dual role in neurodegenerative diseases by causing the downstream effect of promoting the degradation of misfolded proteins and an upstream effect where its deregulation perturbs global proteostasis, contributing to disease progression. Challenges in the future development of therapeutic strategies to target the autophagy pathway are discussed.

  16. Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice.

    Directory of Open Access Journals (Sweden)

    Mianqiu Zhang

    Full Text Available Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7, was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis.

  17. Does bilirubin protect against hemochromatosis gene (HFE) related mortality?

    NARCIS (Netherlands)

    Alizadeh, Behrooz Z.; Njajou, Omer T.; Houwing-Duistermaat, Jeanine J.; de Jong, Gerard; Vergeer, Jeannette M.; Hofman, Albert; Pols, Huibert A.P.; van Duijn, Cornelia M.

    2004-01-01

    Serum bilirubin is an important antioxidant that is found at increased levels in hereditary hemochromatosis patients. We hypothesized that increased levels of serum bilirubin may play a protective role against oxidative stress induced by iron overload in carriers of mutations in the hereditary hemoc

  18. [Protection of corneal endothelium from apoptosis by gene and cell therapy].

    Science.gov (United States)

    Fuchsluger, T A

    2016-06-01

    Protection of corneal endothelium from apoptosis using gene and cell therapy is in a translational phase. This approach offers advantages for eye banking and after transplantation. Safe vehicles for gene or cell therapeutic transduction of corneal endothelium with nucleic acids are available. This strategy will be further developed in consultation with the Paul Ehrlich Institute and European regulatory authorities.

  19. TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis.

    Science.gov (United States)

    Inokuchi-Shimizu, Sayaka; Park, Eek Joong; Roh, Yoon Seok; Yang, Ling; Zhang, Bi; Song, Jingyi; Liang, Shuang; Pimienta, Michael; Taniguchi, Koji; Wu, Xuefeng; Asahina, Kinji; Lagakos, William; Mackey, Mason R; Akira, Shizuo; Ellisman, Mark H; Sears, Dorothy D; Olefsky, Jerrold M; Karin, Michael; Brenner, David A; Seki, Ekihiro

    2014-08-01

    The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

  20. Early autophagy activation inhibits podocytes from apoptosis induced by aldosterone

    Institute of Scientific and Technical Information of China (English)

    王文琰

    2013-01-01

    Objective To explore the protection of early autoph-agy activation on podocyte injury induced by aldosterone.Methods In vitro cultured mouse podocyte clones(MPC5) were treated with aldosterone for 6,12,24,48 hrespectively. Apoptosis of podocytes was detected by

  1. Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

    Science.gov (United States)

    1988-02-02

    Montie, S. Kadis, and S. I. Ajl (ed.), Microbial toxins, vol. 3. Academic Press, Inc., New York. 23. Little, S. F., and G. B. Knudaon. 1986...Takkinen, and L. Kaariainen. 1981. Nucleotide sequence of the promoter and NHa-terminal signal peptide region of the a- amylase gene from Bacillus

  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. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    Science.gov (United States)

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  4. Autophagy: Regulation by Energy Sensing

    NARCIS (Netherlands)

    A.J. Meijer; P. Codogno

    2011-01-01

    Autophagy is inhibited by the mTOR signaling pathway, which is stimulated by increased amino acid levels. When cellular energy production is compromised, AMP-activated protein kinase is activated, mTOR is inhibited and autophagy is stimulated. Two recent studies have shed light on the molecular mech

  5. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    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.

  6. Regulation of Autophagy-Related Protein and Cell Differentiation by High Mobility Group Box 1 Protein in Adipocytes

    Directory of Open Access Journals (Sweden)

    Huanhuan Feng

    2016-01-01

    Full Text Available High mobility group box 1 protein (HMGB1 is a molecule related to the development of inflammation. Autophagy is vital to maintain cellular homeostasis and protect against inflammation of adipocyte injury. Our recent work focused on the relationship of HMGB1 and autophagy in 3T3-L1 cells. In vivo experimental results showed that, compared with the normal-diet group, the high-fat diet mice displayed an increase in adipocyte size in the epididymal adipose tissues. The expression levels of HMGB1 and LC3II also increased in epididymal adipose tissues in high-fat diet group compared to the normal-diet mice. The in vitro results indicated that HMGB1 protein treatment increased LC3II formation in 3T3-L1 preadipocytes in contrast to that in the control group. Furthermore, LC3II formation was inhibited through HMGB1 knockdown by siRNA. Treatment with the HMGB1 protein enhanced LC3II expression after 2 and 4 days but decreased the expression after 8 and 10 days among various differentiation stages of adipocytes. By contrast, FABP4 expression decreased on the fourth day and increased on the eighth day. Hence, the HMGB1 protein modulated autophagy-related proteins and lipid-metabolism-related genes in adipocytes and could be a new target for treatment of obesity and related metabolic diseases.

  7. Neuronal autophagy in cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Feng Xu; Jin-Hua Gu; Zheng-Hong Qin

    2012-01-01

    Autophagy has evolved as a conserved process for the bulk degradation and recycling of cytosolic components,such as long-lived proteins and organelles.In neurons,autophagy is important for homeostasis and protein quality control and is maintained at relatively low levels under normal conditions,while it is upregulated in response to pathophysiological conditions,such as cerebral ischemic injury.However,the role of autophagy is more complex.It depends on age or brain maturity,region,severity of insult,and the stage of ischemia.Whether autophagy plays a beneficial or a detrimental role in cerebral ischemia depends on various pathological conditions.In this review,we elucidate the role of neuronal autophagy in cerebral ischemia.

  8. Impaired autophagy contributes to adverse cardiac remodeling in acute myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Xiaoqian Wu

    Full Text Available OBJECTIVE: Autophagy is activated in ischemic heart diseases, but its dynamics and functional roles remain unclear and controversial. In this study, we investigated the dynamics and role of autophagy and the mechanism(s, if any, during postinfarction cardiac remodeling. METHODS AND RESULTS: Acute myocardial infarction (AMI was induced by ligating left anterior descending (LAD coronary artery. Autophagy was found to be induced sharply 12-24 hours after surgery by testing LC3 modification and Electron microscopy. P62 degradation in the infarct border zone was increased from day 0.5 to day 3, and however, decreased from day 5 until day 21 after LAD ligation. These results indicated that autophagy was induced in the acute phase of AMI, and however, impaired in the latter phase of AMI. To investigate the significance of the impaired autophagy in the latter phase of AMI, we treated the mice with Rapamycin (an autophagy enhancer, 2.0 mg/kg/day or 3-methyladenine (3MA, an autophagy inhibitor, 15 mg/kg/day one day after LAD ligation until the end of experiment. The results showed that Rapamycin attenuated, while 3MA exacerbated, postinfarction cardiac remodeling and dysfunction respectively. In addition, Rapamycin protected the H9C2 cells against oxygen glucose deprivation in vitro. Specifically, we found that Rapamycin attenuated NFκB activation after LAD ligation. And the inflammatory response in the acute stage of AMI was significantly restrained with Rapamycin treatment. In vitro, inhibition of NFκB restored autophagy in a negative reflex. CONCLUSION: Sustained myocardial ischemia impairs cardiomyocyte autophagy, which is an essential mechanism that protects against adverse cardiac remodeling. Augmenting autophagy could be a therapeutic strategy for acute myocardial infarction.

  9. 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.

  10. Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes.

    Science.gov (United States)

    Mei, Shuang; Ni, Hong-Min; Manley, Sharon; Bockus, Abigail; Kassel, Karen M; Luyendyk, James P; Copple, Bryan L; Ding, Wen-Xing

    2011-11-01

    Fatty acid-induced lipotoxicity plays a critical role in the pathogenesis of nonalcoholic liver disease. Saturated fatty acids and unsaturated fatty acids have differential effects on cell death and steatosis, but the mechanisms responsible for these differences are not known. Using cultured HepG2 cells and primary mouse hepatocytes, we found that unsaturated and saturated fatty acids differentially regulate autophagy and apoptosis. The unsaturated fatty acid, oleic acid, promoted the formation of triglyceride-enriched lipid droplets and induced autophagy but had a minimal effect on apoptosis. In contrast, the saturated fatty acid, palmitic acid, was poorly converted into triglyceride-enriched lipid droplets, suppressed autophagy, and significantly induced apoptosis. Subsequent studies revealed that palmitic acid-induced apoptosis suppressed autophagy by inducing caspase-dependent Beclin 1 cleavage, indicating cross-talk between apoptosis and autophagy. Moreover, our data suggest that the formation of triglyceride-enriched lipid droplets and induction of autophagy are protective mechanisms against fatty acid-induced lipotoxicity. In line with our in vitro findings, we found that high-fat diet-induced hepatic steatosis was associated with autophagy in the mouse liver. Potential modulation of autophagy may be a novel approach that has therapeutic benefits for obesity-induced steatosis and liver injury.

  11. MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy.

    Science.gov (United States)

    Pan, Wei; Zhong, Yun; Cheng, Chuanfang; Liu, Benrong; Wang, Li; Li, Aiqun; Xiong, Longgen; Liu, Shiming

    2013-01-01

    Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.

  12. MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy.

    Directory of Open Access Journals (Sweden)

    Wei Pan

    Full Text Available Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group. Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene, and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.

  13. Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy

    Science.gov (United States)

    Cullup, Thomas; Kho, Ay L.; Dionisi-Vici, Carlo; Brandmeier, Birgit; Smith, Frances; Urry, Zoe; Simpson, Michael A.; Yau, Shu; Bertini, Enrico; McClelland, Verity; Al-Owain, Mohammed; Koelker, Stefan; Koerner, Christian; Hoffmann, Georg F.; Wijburg, Frits A.; Hoedt, Amber E. ten; Rogers, Curtis; Manchester, David; Miyata, Rie; Hayashi, Masaharu; Said, Elizabeth; Soler, Doriette; Kroisel, Peter M.; Windpassinger, Christian; Filloux, Francis M.; Al-Kaabi, Salwa; Hertecant, Jozef; Del Campo, Miguel; Buk, Stefan; Bodi, Istvan; Goebel, Hans-Hilmar; Sewry, Caroline A.; Abbs, Stephen; Mohammed, Shehla; Josifova, Dragana; Gautel, Mathias; Jungbluth, Heinz

    2012-01-01

    Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 patients. We identified recessive mutations in EPG5 (previously KIAA1632), indicating a causative role in Vici syndrome. EPG5 is the human homologue of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies demonstrated a severe block of autophagosomal clearance in muscle and fibroblasts from EPG5 mutant patients, resulting in autophagic cargo accumulation in autophagosomes. These findings indicate Vici syndrome as a paradigm of a human multisystem disorder associated with defective autophagy, and suggest a fundamental role of the autophagy pathway in the anatomical and functional formation of organs such as the brain, the heart and the immune system. PMID:23222957

  14. The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis.

    Science.gov (United States)

    Goodall, Megan L; Fitzwalter, Brent E; Zahedi, Shadi; Wu, Min; Rodriguez, Diego; Mulcahy-Levy, Jean M; Green, Douglas R; Morgan, Michael; Cramer, Scott D; Thorburn, Andrew

    2016-05-23

    Although autophagy controls cell death and survival, underlying mechanisms are poorly understood, and it is unknown whether autophagy affects only whether or not cells die or also controls other aspects of programmed cell death. MAP3K7 is a tumor suppressor gene associated with poor disease-free survival in prostate cancer. Here, we report that Map3k7 deletion in mouse prostate cells sensitizes to cell death by TRAIL (TNF-related apoptosis-inducing ligand). Surprisingly, this death occurs primarily through necroptosis, not apoptosis, due to assembly of the necrosome in association with the autophagy machinery, mediated by p62/SQSTM1 recruitment of RIPK1. The mechanism of cell death switches to apoptosis if p62-dependent recruitment of the necrosome to the autophagy machinery is blocked. These data show that the autophagy machinery can control the mechanism of programmed cell death by serving as a scaffold rather than by degrading cargo.

  15. Methylprednisolone exerts neuroprotective effects by regulating autophagy and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Wei Gao; Shu-rui Chen; Meng-yao Wu; Kai Gao; Yuan-long Li; Hong-yu Wang; Chen-yuan Li; Hong Li

    2016-01-01

    Methylprednisolone markedly reduces autophagy and apoptosis after secondary spinal cord injury. Here, we investigated whether pretreat-ment of cells with methylprednisolone would protect neuron-like cells from subsequent oxidative damagevia suppression of autophagy and apoptosis. Cultured N2a cells were pretreated with 10 µM methylprednisolone for 30 minutes, then exposed to 100 µM H2O2 for 24 hours. Inverted phase contrast microscope images, MTT assay, lfow cytometry and western blot results showed that, compared to cells ex-posed to 100 µM H2O2 alone, cells pretreated with methylprednisolone had a signiifcantly lower percentage of apoptotic cells, maintained a healthy morphology, and showed downregulation of autophagic protein light chain 3B and Beclin-1 protein expression. These ifndings indicate that methylprednisolone exerted neuroprotective effects against oxidative damage by suppressing autophagy and apoptosis.

  16. Ammonia Induces Autophagy through Dopamine Receptor D3 and MTOR.

    Science.gov (United States)

    Li, Zhiyuan; Ji, Xinmiao; Wang, Wenchao; Liu, Juanjuan; Liang, Xiaofei; Wu, Hong; Liu, Jing; Eggert, Ulrike S; Liu, Qingsong; Zhang, Xin

    2016-01-01

    Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors.

  17. Boosting autophagy in the diabetic heart: a translational perspective.

    Science.gov (United States)

    Sciarretta, Sebastiano; Boppana, V Subbarao; Umapathi, Mahaa; Frati, Giacomo; Sadoshima, Junichi

    2015-10-01

    Diabetes, obesity, and dyslipidemia are main risk factors that promote the development of cardiovascular diseases. These metabolic abnormalities are frequently found to be associated together in a highly morbid clinical condition called metabolic syndrome. Metabolic derangements promote endothelial dysfunction, atherosclerotic plaque formation and rupture, cardiac remodeling and dysfunction. This evidence strongly encourages the elucidation of the mechanisms through which obesity, diabetes, and metabolic syndrome induce cellular abnormalities and dysfunction in order to discover new therapeutic targets and strategies for their prevention and treatment. Numerous studies employing both dietary and genetic animal models of obesity and diabetes have demonstrated that autophagy, an intracellular system for protein degradation, is impaired in the heart under these conditions. This suggests that autophagy reactivation may represent a future potential therapeutic intervention to reduce cardiac maladaptive alterations in patients with metabolic derangements. In fact, autophagy is a critical mechanism to preserve cellular homeostasis and survival. In addition, the physiological activation of autophagy protects the heart during stress, such as acute ischemia, starvation, chronic myocardial infarction, pressure overload, and proteotoxic stress. All these aspects will be discussed in our review article together with the potential ways to reactivate autophagy in the context of obesity, metabolic syndrome, and diabetes.

  18. Autophagy mediates tolerance to Staphylococcus aureus alpha-toxin.

    Science.gov (United States)

    Maurer, Katie; Reyes-Robles, Tamara; Alonzo, Francis; Durbin, Joan; Torres, Victor J; Cadwell, Ken

    2015-04-01

    Resistance and tolerance are two defense strategies employed by the host against microbial threats. Autophagy-mediated degradation of bacteria has been extensively described as a major resistance mechanism. Here we find that the dominant function of autophagy proteins during infections with the epidemic community-associated methicillin-resistant Staphylococcus aureus USA300 is to mediate tolerance rather than resistance. Atg16L1 hypomorphic mice (Atg16L1(HM)), which have reduced autophagy, were highly susceptible to lethality in both sepsis and pneumonia models of USA300 infection. Autophagy confers protection by limiting the damage caused by α-toxin, particularly to endothelial cells. Remarkably, Atg16L1(HM) mice display enhanced survival rather than susceptibility upon infection with α-toxin-deficient S. aureus. These results identify an essential role for autophagy in tolerance to Staphylococcal disease and highlight how a single virulence factor encoded by a pathogen can determine whether a given host factor promotes tolerance or resistance.

  19. Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana.

    Science.gov (United States)

    Chen, Liang; Liao, Bin; Qi, Hua; Xie, Li-Juan; Huang, Li; Tan, Wei-Juan; Zhai, Ning; Yuan, Li-Bing; Zhou, Ying; Yu, Lu-Jun; Chen, Qin-Fang; Shu, Wensheng; Xiao, Shi

    2015-01-01

    Autophagy involves massive degradation of intracellular components and functions as a conserved system that helps cells to adapt to adverse conditions. In mammals, hypoxia rapidly stimulates autophagy as a cell survival response. Here, we examine the function of autophagy in the regulation of the plant response to submergence, an abiotic stress that leads to hypoxia and anaerobic respiration in plant cells. In Arabidopsis thaliana, submergence induces the transcription of autophagy-related (ATG) genes and the formation of autophagosomes. Consistent with this, the autophagy-defective (atg) mutants are hypersensitive to submergence stress and treatment with ethanol, the end product of anaerobic respiration. Upon submergence, the atg mutants have increased levels of transcripts of anaerobic respiration genes (alcohol dehydrogenase 1, ADH1 and pyruvate decarboxylase 1, PDC1), but reduced levels of transcripts of other hypoxia- and ethylene-responsive genes. Both submergence and ethanol treatments induce the accumulation of reactive oxygen species (ROS) in the rosettes of atg mutants more than in the wild type. Moreover, the production of ROS by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases is necessary for plant tolerance to submergence and ethanol, submergence-induced expression of ADH1 and PDC1, and activation of autophagy. The submergence- and ethanol-sensitive phenotypes in the atg mutants depend on a complete salicylic acid (SA) signaling pathway. Together, our findings demonstrate that submergence-induced autophagy functions in the hypoxia response in Arabidopsis by modulating SA-mediated cellular homeostasis.

  20. 细胞自噬与肿瘤耐药的研究进展%Recent progress of autophagy and drug-resistance

    Institute of Scientific and Technical Information of China (English)

    叶丽霖; 李玉峰

    2013-01-01

    Summary Autophagy is a self degradation process that are responsible for the removal of long lived proteins and damaged organelles, which plays a key role in the elimination of such intracellular waste, reconstruction of structure,growth and development of cells and maintainance of cellular homeostasis. During tumour development, autophagy has paradoxically been reported to have roles in promoting both cell survival and growth. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation,and to mitigate genome damage in tumour cells in response to metabolic stress. Many studies show that many cytoxic drugs can induce autophagy in its induction of apoptosis. Activation of autophagy can lead to drug resistance in tumour cells. In this review, we will summarize the relationship between autophagy and drug resistance.

  1. microRNA-101 is a potent inhibitor of autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Wen, Jiayu; Lees, Michael

    2011-01-01

    performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we...... identified three novel miR-101 targets, STMN1, RAB5A and ATG4D. siRNA-mediated depletion of these genes phenocopied the effect of miR-101 overexpression, demonstrating their importance in autophagy regulation. Importantly, overexpression of STMN1 could partially rescue cells from miR-101-mediated inhibition...

  2. A role of autophagy in PTP4A3-driven cancer progression.

    Science.gov (United States)

    Huang, Yu-Han; Al-Aidaroos, Abdul Qader O; Yuen, Hiu-Fung; Zhang, Shu-Dong; Shen, Han-Ming; Rozycka, Ewelina; McCrudden, Cian M; Tergaonkar, Vinay; Gupta, Abhishek; Lin, You Bin; Thiery, Jean Paul; Murray, James T; Zeng, Qi

    2014-10-01

    Autophagy, a "self-eating" cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.

  3. 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.

  4. 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.

  5. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.

    Science.gov (United States)

    Sinha, Rohit Anthony; You, Seo-Hee; Zhou, Jin; Siddique, Mobin M; Bay, Boon-Huat; Zhu, Xuguang; Privalsky, Martin L; Cheng, Sheue-Yann; Stevens, Robert D; Summers, Scott A; Newgard, Christopher B; Lazar, Mitchell A; Yen, Paul M

    2012-07-01

    For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

  6. Autophagy is required for the activation of NFκB.

    Science.gov (United States)

    Criollo, Alfredo; Chereau, Fanny; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Mariño, Guillermo; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Baud, Véronique; Kroemer, Guido

    2012-01-01

    It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.

  7. Mx1 gene protects mice against the highly lethal human H5N1 influenza virus.

    Science.gov (United States)

    Salomon, Rachelle; Staeheli, Peter; Kochs, Georg; Yen, Hui-Ling; Franks, John; Rehg, Jerold E; Webster, Robert G; Hoffmann, Erich

    2007-10-01

    We investigated the importance of the host Mx1 gene in protection against highly pathogenic H5N1 avian influenza virus. Mice expressing the Mx1 gene survived infection with the lethal human H5N1 isolate A/Vietnam/1203/04 and with reassortants combining its genes with those of the non-lethal virus A/chicken/Vietnam/C58/04, while all Mx1-/- mice succumbed. Mx1-expressing mice showed lower organ virus titers, fewer lesions, and less pulmonary inflammation. Our data support the hypothesis that Mx1 expression protects mice against the high pathogenicity of H5N1 virus through inhibition of viral polymerase activity ultimately resulting in reduced viral growth and spread. Drugs that mimic this mechanism may be protective in humans.

  8. 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.

  9. Gene Transfer to Dendritic Cells Induced a Protective Immunity against Melanoma

    Institute of Scientific and Technical Information of China (English)

    Pat Metharom; Kay A.O. Ellem; Ming Q. Wei

    2005-01-01

    Lentiviral vectors have shown promises for efficient gene transfer to dividing as well as nondividing cells. In this study, we explored lentiviral vector-mediated, the entire mTRP-2 gene transfer and expression in dendritic cells (DCs). Adoptive transfer of DCs-expressing mTRP-2 (DC-HR'CmT2) into C57BL/6 mouse was also assessed.Dendritic cells were harvested from bone marrow and functional DCs were proved by allogeneic mixed lymphocyte reaction. Lentiviral vectors were produced by transient transfection of 293T cells. Transduction of DCs was proved by marker gene expression and PCR and RT-PCR amplification. Implantation of the transduced DCs, depletion of immune cells as well as the survival of the mice after tumour challenge were investigated. High efficiency of gene transfer into mature DCs was achieved. The high level expression of the functional antigen (TRP-2) and induction of protective immunity by adoptive transfer of TRP-2 gene modified DCs were demonstrated. In vivo study showed a complete protection of mice from further melanoma cell challenge. In comparison, only 83% of mice survived when mTRP-2 peptide-pulsed DCs were administered, suggesting the generation of specific protection. Together, these results demonstrated the usefulness of this gene transfer to DC approach for immunotherapy of cancer and indicated that using tumour associated antigens (TAAs) for gene transfer may be potentially beneficial for the therapy of melanoma.

  10. 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.

  11. Transcriptional and epigenetic regulation of autophagy in aging.

    Science.gov (United States)

    Lapierre, Louis R; Kumsta, Caroline; Sandri, Marco; Ballabio, Andrea; Hansen, Malene

    2015-01-01

    Macroautophagy is a major intracellular degradation process recognized as playing a central role in cell survival and longevity. This multistep process is extensively regulated at several levels, including post-translationally through the action of conserved longevity factors such as the nutrient sensor TOR. More recently, transcriptional regulation of autophagy genes has emerged as an important mechanism for ensuring the somatic maintenance and homeostasis necessary for a long life span. Autophagy is increased in many long-lived model organisms and contributes significantly to their longevity. In turn, conserved transcription factors, particularly the helix-loop-helix transcription factor TFEB and the forkhead transcription factor FOXO, control the expression of many autophagy-related genes and are important for life-span extension. In this review, we discuss recent progress in understanding the contribution of these transcription factors to macroautophagy regulation in the context of aging. We also review current research on epigenetic changes, such as histone modification by the deacetylase SIRT1, that influence autophagy-related gene expression and additionally affect aging. Understanding the molecular regulation of macroautophagy in relation to aging may offer new avenues for the treatment of age-related diseases.

  12. Elaborating the role of natural products-induced autophagy in cancer treatment: achievements and artifacts in the state of the art.

    Science.gov (United States)

    Wang, Ning; Feng, Yibin

    2015-01-01

    Autophagy is a homeostatic process that is highly conserved across different types of mammalian cells. Autophagy is able to relieve tumor cell from nutrient and oxidative stress during the rapid expansion of cancer. Excessive and sustained autophagy may lead to cell death and tumor shrinkage. It was shown in literature that many anticancer natural compounds and extracts could initiate autophagy in tumor cells. As summarized in this review, the tumor suppressive action of natural products-induced autophagy may lead to cell senescence, provoke apoptosis-independent cell death, and complement apoptotic cell death by robust or target-specific mechanisms. In some cases, natural products-induced autophagy could protect tumor cells from apoptotic death. Technical variations in detecting autophagy affect data quality, and study focus should be made on elaborating the role of autophagy in deciding cell fate. In vivo study monitoring of autophagy in cancer treatment is expected to be the future direction. The clinical-relevant action of autophagy-inducing natural products should be highlighted in future study. As natural products are an important resource in discovery of lead compound of anticancer drug, study on the role of autophagy in tumor suppressive effect of natural products continues to be necessary and emerging.

  13. Autophagy is required for G₁/G₀ quiescence in response to nitrogen starvation in Saccharomyces cerevisiae.

    Science.gov (United States)

    An, Zhenyi; Tassa, Amina; Thomas, Collin; Zhong, Rui; Xiao, Guanghua; Fotedar, Rati; Tu, Benjamin P; Klionsky, Daniel J; Levine, Beth

    2014-10-01

    In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G 0₀ autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.

  14. 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.

  15. Paradoxical role of autophagy in the dysplastic and tumor-forming stages of hepatocarcinoma development in rats.

    Science.gov (United States)

    Sun, K; Guo, X-L; Zhao, Q-d; Jing, Y-y; Kou, X-r; Xie, X-q; Zhou, Y; Cai, N; Gao, L; Zhao, X; Zhang, S-s; Song, J-r; Li, D; Deng, W-j; Li, R; Wu, M-c; Wei, L-x

    2013-02-21

    Many reports have shown that autophagy has a role as both a promoter and inhibitor in tumor development. However, the mechanism of this paradox is unknown. Tumor development is a multistep process. Therefore, we investigated whether the role of autophagy in hepatocarcinoma formation depended on the stage of tumor development. Based on our results, autophagy inhibition by chloroquine had a tumor-promotive effect in the rat model with N-diethylnitrosamine-induced hepatocarcinogenesis in its dysplastic stage (Ds) and a tumor-suppressive effect in its tumor-forming stage (Ts). In the Ds, autophagy inhibition enhanced cell proliferation, DNA damage and inflammatory cytokines expression in liver. These changes were dependent on the upregulation of reactive oxygen species (ROS) that was resulted from autophagy inhibition, and ultimately accelerated the process of hepatocarcinogenesis. However, in the Ts, autophagy inhibition restrained tumor formation by decreasing tumor cell survival and proliferation. In this stage, autophagy inhibition led to excessive ROS accumulation in the tumor, which promoted cell apoptosis, and prominently suppressed tumor cell metabolism. Taken together, our data suggested that autophagy suppressed hepatocarcinogenesis in the Ds by protecting normal cell stability and promoted hepatocarcinogenesis in the Ts by supporting tumor cells growth. Autophagy always had a role as a protector throughout the process of hepatocarcinoma development.

  16. Defective Autophagy Initiates Malignant Transformation.

    Science.gov (United States)

    Galluzzi, Lorenzo; Bravo-San Pedro, José Manuel; Kroemer, Guido

    2016-05-19

    In this issue of Molecular Cell, Park et al. (2016) elegantly demonstrate that a partial defect in autophagy supports malignant transformation as it favors the production of genotoxic reactive oxygen species by mitochondria.

  17. Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

    Institute of Scientific and Technical Information of China (English)

    Xiao-hong LIU; Fu-cheng LIN

    2008-01-01

    Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.

  18. Role of autophagy in COPD skeletal muscle dysfunction.

    Science.gov (United States)

    Hussain, Sabah N A; Sandri, Marco

    2013-05-01

    Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by parenchymal damage and irreversible airflow limitation. In addition to lung dysfunction, patients with COPD develop weight loss, malnutrition, poor exercise performance, and skeletal muscle atrophy. The latter has been attributed to an imbalance between muscle protein synthesis and protein degradation. Several reports have confirmed that enhanced protein degradation and atrophy of limb muscles of COPD patient is mediated in part through activation of the ubiquitin-proteasome pathway and that this activation is triggered by enhanced production of reactive oxygen species. Until recently, the importance of the autophagy-lysosome pathway in protein degradation of skeletal muscles has been largely ignored, however, recent evidence suggests that this pathway is actively involved in recycling of cytosolic proteins, organelles, and protein aggregates in normal skeletal muscles. The protective role of autophagy in the regulation of muscle mass has recently been uncovered in mice with muscle-specific suppression of autophagy. These mice develop severe muscle weakness, atrophy, and decreased muscle contractility. No information is yet available about the involvement of the autophagy in the regulation of skeletal muscle mass in COPD patients. Pilot experiments on vastus lateralis muscle samples suggest that the autophagy-lysosome system is induced in COPD patients compared with control subjects. In this review, we summarize recent progress related to molecular structure, regulation, and roles of the autophagy-lysosome pathway in normal and diseased skeletal muscles. We also speculate about regulation and functional importance of this system in skeletal muscle dysfunction in COPD patients.

  19. 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.

  20. 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.

  1. 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.

  2. Autophagy and access: understanding the role of androgen receptor subcellular localization in SBMA.

    Science.gov (United States)

    Montie, Heather L; Merry, Diane E

    2009-11-01

    Ridding neurons of toxic misfolded proteins is a critical feature of many neurodegenerative diseases. We have recently reported that lack of access of nuclear polyglutamine-expanded androgen receptor (AR) to the autophagic degradation pathway is a critical point in pathogenesis. When mutant AR is contained within the cytoplasm, it can be degraded by autophagy, resulting in amelioration of its toxic effects, as has been observed in other polyglutamine expansion diseases involving cytoplasmic mutant proteins. However, we have also found that pharmacological induction of autophagy protects SBMA motor neurons from the toxic effects of even nuclear localized mutant AR, albeit without affecting mutant nuclear AR levels. Thus, we have further investigated the mechanism by which autophagy elicits therapeutic benefit in cell culture. We found that endogenous autophagy only slightly alters nuclear mutant AR aggregation compared to substantial effects on cytoplasmic AR aggregation. Interestingly, pharmacological activation of mTOR-dependent autophagy did not significantly alter nuclear AR aggregation, whereas we observed that it protects SBMA motor neurons. Our findings indicate that therapeutic intervention to induce autophagy represents a potential potent benefit for SBMA, and that it likely does so by protecting SBMA motor neurons independent of a direct effect on mutant AR.

  3. Role and regulation of autophagy in heat stress responses of tomato plants.

    Science.gov (United States)

    Zhou, Jie; Wang, Jian; Yu, Jing-Quan; Chen, Zhixiang

    2014-01-01

    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 (VIGS) 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.

  4. 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.

  5. Elastase induces lung epithelial cell autophagy through placental growth factor

    Science.gov (United States)

    Hou, Hsin-Han; Cheng, Shih-Lung; Chung, Kuei-Pin; Kuo, Mark Yen-Ping; Yeh, Cheng-Chang; Chang, Bei-En; Lu, Hsuan-Hsuan; Wang, Hao-Chien; Yu, Chong-Jen

    2014-01-01

    Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD. PMID:24988221

  6. Macrophage Autophagy in Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Maria Chiara Maiuri

    2013-01-01

    Full Text Available Macrophages play crucial roles in atherosclerotic immune responses. Recent investigation into macrophage autophagy (AP in atherosclerosis has demonstrated a novel pathway through which these cells contribute to vascular inflammation. AP is a cellular catabolic process involving the delivery of cytoplasmic contents to the lysosomal machinery for ultimate degradation and recycling. Basal levels of macrophage AP play an essential role in atheroprotection during early atherosclerosis. However, AP becomes dysfunctional in the more advanced stages of the pathology and its deficiency promotes vascular inflammation, oxidative stress, and plaque necrosis. In this paper, we will discuss the role of macrophages and AP in atherosclerosis and the emerging evidence demonstrating the contribution of macrophage AP to vascular pathology. Finally, we will discuss how AP could be targeted for therapeutic utility.

  7. IL13 activates autophagy to regulate secretion in airway epithelial cells.

    Science.gov (United States)

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

  8. Berberine alleviates cardiac ischemia/reperfusion injury by inhibiting excessive autophagy in cardiomyocytes.

    Science.gov (United States)

    Huang, Zhouqing; Han, Zhihua; Ye, Bozhi; Dai, Zhenyu; Shan, Peiren; Lu, Zhongqiu; Dai, Kezhi; Wang, Changqian; Huang, Weijian

    2015-09-05

    Ischemia/reperfusion (I/R)-induced autophagy increases the severity of cardiomyocyte injury. The aim of this study was to investigate the effects of berberine, a natural extract from Rhizoma coptidis, on the I/R-induced excessive autophagy in in vitro and in vivo models. Autophagy was increased both in H9c2 myocytes during hypoxia/reoxygenation (H/R) injury and in mouse hearts exposed to I/R. And the expression level of p-AMPK and p-mTORC2 (Ser2481) were increased during H/R period. In addition, the increased autophagy level was correlated with reduced cell survival in H9c2 myocytes and increased infarct size in mouse hearts. However, berberine treatment significantly enhanced the H/R-induced cell viability and reduced I/R-induced myocardial infarct size, which was accompanied by improved cardiac function. The beneficial effect of berberine is associated with inhibiting the cellular autophagy level, due to decreasing the expression level of autophagy-related proteins such as SIRT1, BNIP3, and Beclin-1. Furthermore, both the level of p-AMPK and p-mTORC2 (Ser2481) in H9c2 myocytes exposed to H/R were decreased by berberine. In summary, berberine protects myocytes during I/R injury through suppressing autophagy activation. Therefore, berberine may be a promising agent for treating I/R-induced cardiac myocyte injury.

  9. Cross-cancer profiling of molecular alterations within the human autophagy interaction network.

    Science.gov (United States)

    Lebovitz, Chandra B; Robertson, A Gordon; Goya, Rodrigo; Jones, Steven J; Morin, Ryan D; Marra, Marco A; Gorski, Sharon M

    2015-01-01

    Aberrant activation or disruption of autophagy promotes tumorigenesis in various preclinical models of cancer, but whether the autophagy pathway is a target for recurrent molecular alteration in human cancer patient samples is unknown. To address this outstanding question, we surveyed 211 human autophagy-associated genes for tumor-related alterations to DNA sequence and RNA expression levels and examined their association with patient survival outcomes in multiple cancer types with sequence data from The Cancer Genome Atlas consortium. We found 3 (RB1CC1/FIP200, ULK4, WDR45/WIPI4) and one (ATG7) core autophagy genes to be under positive selection for somatic mutations in endometrial carcinoma and clear cell renal carcinoma, respectively, while 29 autophagy regulators and pathway interactors, including previously identified KEAP1, NFE2L2, and MTOR, were significantly mutated in 6 of the 11 cancer types examined. Gene expression analyses revealed that GABARAPL1 and MAP1LC3C/LC3C transcripts were less abundant in breast cancer and non-small cell lung cancers than in matched normal tissue controls; ATG4D transcripts were increased in lung squamous cell carcinoma, as were ATG16L2 transcripts in kidney cancer. Unsupervised clustering of autophagy-associated mRNA levels in tumors stratified patient overall survival in 3 of 9 cancer types (acute myeloid leukemia, clear cell renal carcinoma, and head and neck cancer). These analyses provide the first comprehensive resource of recurrently altered autophagy-associated genes in human tumors, and highlight cancer types and subtypes where perturbed autophagy may be relevant to patient overall survival.

  10. 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.

  11. Interactions between Autophagy and Inhibitory Cytokines.

    Science.gov (United States)

    Wu, Tian-Tian; Li, Wei-Min; Yao, Yong-Ming

    2016-01-01

    Autophagy is a degradative pathway that plays an essential role in maintaining cellular homeostasis. Most early studies of autophagy focused on its involvement in age-associated degeneration and nutrient deprivation. However, the immunological functions of autophagy have become more widely studied in recent years. Autophagy has been shown to be an intrinsic cellular defense mechanism in the innate and adaptive immune responses. Cytokines belong to a broad and loose category of proteins and are crucial for innate and adaptive immunity. Inhibitory cytokines have evolved to permit tolerance to self while also contributing to the eradication of invading pathogens. Interactions between inhibitory cytokines and autophagy have recently been reported, revealing a novel mechanism by which autophagy controls the immune response. In this review, we discuss interactions between autophagy and the regulatory cytokines IL-10, transforming growth factor-β, and IL-27. We also mention possible interactions between two newly discovered cytokines, IL-35 and IL-37, and autophagy.

  12. 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.

  13. Interactions between Autophagy and Inhibitory Cytokines

    Science.gov (United States)

    Wu, Tian-tian; Li, Wei-Min; Yao, Yong-Ming

    2016-01-01

    Autophagy is a degradative pathway that plays an essential role in maintaining cellular homeostasis. Most early studies of autophagy focused on its involvement in age-associated degeneration and nutrient deprivation. However, the immunological functions of autophagy have become more widely studied in recent years. Autophagy has been shown to be an intrinsic cellular defense mechanism in the innate and adaptive immune responses. Cytokines belong to a broad and loose category of proteins and are crucial for innate and adaptive immunity. Inhibitory cytokines have evolved to permit tolerance to self while also contributing to the eradication of invading pathogens. Interactions between inhibitory cytokines and autophagy have recently been reported, revealing a novel mechanism by which autophagy controls the immune response. In this review, we discuss interactions between autophagy and the regulatory cytokines IL-10, transforming growth factor-β, and IL-27. We also mention possible interactions between two newly discovered cytokines, IL-35 and IL-37, and autophagy. PMID:27313501

  14. Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy.

    Science.gov (United States)

    Papadakis, Michalis; Hadley, Gina; Xilouri, Maria; Hoyte, Lisa C; Nagel, Simon; McMenamin, M Mary; Tsaknakis, Grigorios; Watt, Suzanne M; Drakesmith, Cynthia Wright; Chen, Ruoli; Wood, Matthew J A; Zhao, Zonghang; Kessler, Benedikt; Vekrellis, Kostas; Buchan, Alastair M

    2013-03-01

    Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

  15. 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.

  16. Calpain2调节自噬相关基因ATG7的表达在非酒精性脂肪性肝病中的作用%Role of regulation of autophagy related gene 7 by Calpain 2 in non-alcoholic fatty liver disease in rats

    Institute of Scientific and Technical Information of China (English)

    陈洁; 熊吉; 陈潇迪; 牟歌; 王军; 樊丽琳; 陈东风

    2011-01-01

    Objective To investigate the expression and significance of autophagy related gene 7 ( ATG7 ) and Calpain 2 in nonalcoholic fatty liver disease ( NAFLD ). Methods In vivo model of NAFLD was established in SD rats by high fat diet, while the rats fed with normal food were set as control group. The rats were killed at 4, 8, 12 and 16 weeks after feeding. Blood samples were collected to check serum aspartate transaminase (AST) , alanine aminotransferase (ALT) , and free fatty acid(FFA). Steatosis of liver tissues were observed by HE staining. The expression of Calpain 2 and ATG7 was detected by real-time PCR and Western blotting respectively for mRNA and protein levels. Results HE staining implicated that the degree of hepatic steatosis was increased with the time of high fat diet feeding. Compared with the control group, the serum contents of ALT, AST, and FFA in NAFLD rats were increased with different degree, and significantly increased at 16th week (165.95 ±7. 24 U/L, 249. 52 ±4. 20 U/L, 0. 83 ±0. 05 mmol/L, respectively P < · 0. 01). The relative expression of Calpain 2 at mRNA level was increased after high fat diet feeding and reached its peak at the 16th week, and was 9. 83 ±0. 85 fold higher as compared with the control group (P <0. 01). While the relative expression of ATG7 began to decrease at the 4th week (0. 82 ±0. 02) , and reached its lowest level at the 16th week (0. 20 ±0. 03, P <0. 01) when compared with the control group. As with mRNA level, the protein level of Calpain 2 began to increase at the 4th week (2. 32 ± 0.45 ) , and was 9. 87 ± 1. 20 fold higher as the control group (P <0. 01). While the expression of ATG7 at protein level was decreased with progress of steatosis, and was significantly reduced at 16th week (0.18 ±0.05, P<0.01). Conclusion The up-regulation of Calpain 2 inhibits the expression of ATG7, which further attenuates the cell protection through autophagy, and then induces injury of hepatocyte in NAFLD. Autophagy may

  17. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    Science.gov (United States)

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy.

  18. Candida albicans autophagy, no longer a bystander: Its role in tolerance to ER stress-related antifungal drugs.

    Science.gov (United States)

    Yu, Qilin; Jia, Chang; Dong, Yijie; Zhang, Bing; Xiao, Chenpeng; Chen, Yulu; Wang, Yuzhou; Li, Xiaoling; Wang, Lei; Zhang, Biao; Li, Mingchun

    2015-08-01

    Autophagy is a degradation process involved in pathogenicity of many pathogenic fungi. However, its roles in Candida albicans, the leading fungal pathogen in human beings, remain to be detailed. Most recently, we found that endoplasmic reticulum (ER) stress-inducing conditions led to transcriptional up-regulation of C. albicans autophagy-related (ATG) genes, implying a possible link between autophagy and ER stress response in this pathogen. Using a series of C. albicans ATG mutants and autophagy reporting systems, we found that both treatment of ER stress-related drugs and loss of the ER calcium pump Spf1 promoted autophagic flux of Atg8 and Lap41 (a homologue of Saccharomyces cerevisiae Ape1), indicating that these conditions induce autophagy. Moreover, deletion of ATG genes in the spf1Δ/Δ mutant rendered cells hypersensitive to these drugs and caused activation of UPR, revealing a role of autophagy in alleviating ER stress. In addition, only treatment of tunicamycin and loss of Spf1 in combination increased autophagic flux of the ER component Sec63, suggesting that most of the ER stress-related conditions cause non-selective autophagy rather than selective ER phagy. This study uncovers the important role of C. albicans autophagy in ER stress response and tolerance to antifungal drugs.

  19. A curated census of autophagy-modulating proteins and small molecules: candidate targets for cancer therapy.

    Science.gov (United States)

    Lorenzi, Philip L; Claerhout, Sofie; Mills, Gordon B; Weinstein, John N

    2014-07-01

    Autophagy, a programmed process in which cell contents are delivered to lysosomes for degradation, appears to have both tumor-suppressive and tumor-promoting functions; both stimulation and inhibition of autophagy have been reported to induce cancer cell death, and particular genes and proteins have been associated both positively and negatively with autophagy. To provide a basis for incisive analysis of those complexities and ambiguities and to guide development of new autophagy-targeted treatments for cancer, we have compiled a comprehensive, curated inventory of autophagy modulators by integrating information from published siRNA screens, multiple pathway analysis algorithms, and extensive, manually curated text-mining of the literature. The resulting inventory includes 739 proteins and 385 chemicals (including drugs, small molecules, and metabolites). Because autophagy is still at an early stage of investigation, we provide extensive analysis of our sources of information and their complex relationships with each other. We conclude with a discussion of novel strategies that could potentially be used to target autophagy for cancer therapy.

  20. Critical role for IL-18 in spontaneous lung inflammation caused by autophagy deficiency.

    Science.gov (United States)

    Abdel Fattah, Elmoataz; Bhattacharya, Abhisek; Herron, Alan; Safdar, Zeenat; Eissa, N Tony

    2015-06-01

    Autophagy is an important component of the immune response. However, the functions of autophagy in human diseases are much less understood. We studied biological consequences of autophagy deficiency in mice lacking the essential autophagy gene Atg7 or Atg5 in myeloid cells. Surprisingly, these mice presented with spontaneous sterile lung inflammation, characterized by marked recruitment of inflammatory cells, submucosal thickening, goblet cell metaplasia, and increased collagen content. Lung inflammation was associated with increase in several proinflammatory cytokines in the bronchoalveolar lavage and in serum. This inflammation was largely driven by IL-18 as a result of constitutive inflammasome activation. Following i.p. LPS injection, autophagy-deficient mice had higher levels of proinflammatory cytokines in lungs and in serum, as well as increased mortality, than control mice. Intranasal bleomycin challenge exacerbated lung inflammation in autophagy-deficient mice and produced more severe fibrotic changes than in control mice. These results uncover a new and important role for autophagy as negative regulator of lung inflammation.

  1. Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

    Science.gov (United States)

    Matsuhara, Hirotada; Yamamoto, Ayumu

    2016-01-01

    Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis.

  2. 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.

  3. 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.

  4. 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

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

    Directory of Open Access Journals (Sweden)

    Hong-Feng Gu

    Full Text Available 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.

  6. Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

    Institute of Scientific and Technical Information of China (English)

    XIONG Ying; YUAN Yuan; JIA Min; YU Bing; HUANG Hanju

    2007-01-01

    To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein.Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

  7. Effect of autophagy induced by dexamethasone on senescence in chondrocytes

    Science.gov (United States)

    Xue, Enxing; Zhang, Yu; Song, Bing; Xiao, Jun; Shi, Zhanjun

    2016-01-01

    The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague-Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein-red fluorescent protein-light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway-associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence-associated (SA)-β-galactosidase (β-gal) staining. A dose-dependent increase in the number of autophagic vacuoles was observed in the DXM-treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose-dependent increase in autophagosome formation was observed in the DXM-treated chondrocytes. Expression of LC3-II and beclin-1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA-β-gal staining indicated that DXM increased cell senescence. Notably, DXM-induced cell senescence was exacerbated by the autophagic inhibitor 3-MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM-induced autophagy. PMID:27572674

  8. Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment

    Institute of Scientific and Technical Information of China (English)

    Kai; Dong; Zi-Cheng; Zhu; Feng-Hua; Wang; Gen-Jie; Ke; Zhang; Yu; Xun; Xu

    2014-01-01

    AIM:To investigate whether photoreceptor necroptosis induced by z-VAD-FMK(pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment.METHODS:Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1.Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8antibody.RESULTS:It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase(RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptornecroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction.CONCLUSION:These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.

  9. 阿米洛利通过自噬-溶酶体途径保护PC12细胞%Amiloride protects PC12 cells from MPP + induced injury via autophagy-lysosome pathway

    Institute of Scientific and Technical Information of China (English)

    张润娉; 李建平

    2014-01-01

    Objective:To evaluate the neuroprotective effects of amiloride; a non-selective blockers of acid-sensing ion channels,on PC12 cells and to view the influence of autophagy-lysosome pathway (ALP).Methods:PC12 cells were tested with methy-phenylpyridi(MPP+),while giving amiloride as interventiong:The cells viability was analyzed by MTT assay; The cells injury was assessed by lactate dehydrogenase (LDH) assay;Flow cytometry was used to study the apoptotic;Western blot analysis was used to study the autophagic mechanisms.Results:MPP + medium treatment resulted in significantly lower survival rate,the supernatant of LDH leakage was significantly higher,the apoptosis rate increased,the expression of LC3-Ⅱ was inhibited while induced more expression of LAMP2a ; but amiloride effectively improved the cell survival rate,reduced LDH leakage rate,inhibited apoptosis,and upregulated LC3-Ⅱ protein,which is associated decrease expression of LAMP2a.Conclusion:Amiloride can protect PC12 cells against MPP +-induced cell death by autophagic,through inhibition of acid-sensing ion channels activity.%目的:研究酸敏感离子通道阻断剂阿米洛利对大鼠肾上腺嗜铬细胞瘤PC12细胞株的保护作用及其对自噬-溶酶体通路的影响.方法:在PC12细胞模型上,观察甲基苯基吡啶(methy-phenylpyridi,MPP+)处理对细胞存活率(MTT检测)、乳酸脱氢酶(LDH)漏出率、细胞凋亡率的影响,并观察阿米洛利对MPP+所致细胞死亡的影响;蛋白质印迹法检测大自噬通路标志蛋白LC3和分子伴侣介导的自噬通路标志蛋白LAMP2a表达水平的变化.结果:MPP+导致细胞存活率明显降低,上清液中LDH漏出率明显升高,细胞凋亡率升高,抑制大自噬通路标志蛋白LC3-Ⅱ的表达,同时上调了分子伴侣介导的自噬通路标志性蛋白LAMP2a的表达水平;而阿米洛利可提高细胞存活率,减少上清液中LDH漏出率,降低细胞凋亡率,并能在激活大自噬通路的同时下调分

  10. 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.

  11. Study on the Mechanism of mTOR-Mediated Autophagy during Electroacupuncture Pretreatment against Cerebral Ischemic Injury

    Science.gov (United States)

    Wu, Zhou-Quan; Cui, Su-yang; Zhu, Liang

    2016-01-01

    This study is aimed at investigating the association between the electroacupuncture (EA) pretreatment-induced protective effect against early cerebral ischemic injury and autophagy. EA pretreatment can protect cerebral ischemic and reperfusion injuries, but whether the attenuation of early cerebral ischemic injury by EA pretreatment was associated with autophagy is not yet clear. This study used the middle cerebral artery occlusion model to monitor the process of ischemic injury. For rats in the EA pretreatment group, EA pretreatment was conducted at Baihui acupoint before ischemia for 30 min for 5 consecutive days. The results suggested that EA pretreatment significantly increased the expression of autophagy in the cerebral cortical area on the ischemic side of rats. But the EA pretreatment-induced protective effects on the brain could be reversed by the specific inhibitor 3-methyladenine of autophagy. Additionally, the Pearson correlation analysis indicated that the impact of EA pretreatment on p-mTOR (2481) was negatively correlated with its impact on autophagy. In conclusion, the mechanism of EA pretreatment at Baihui acupoint against cerebral ischemic injury is mainly associated with the upregulation of autophagy expression, and its regulation of autophagy may depend on mTOR-mediated signaling pathways. PMID:27547233

  12. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression.

    Science.gov (United States)

    Leventhal, Jeremy S; Ni, Jie; Osmond, Morgan; Lee, Kyung; Gusella, G Luca; Salem, Fadi; Ross, Michael J

    2016-01-01

    Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.

  13. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression.

    Directory of Open Access Journals (Sweden)

    Jeremy S Leventhal

    Full Text Available Sepsis related acute kidney injury (AKI is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS, a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO. Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3 and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.

  14. Establishing a novel C.elegans model to investigate the role of autophagy in amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    Jia LI; Kai-xing HUANG; Wei-dong LE

    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 genes in daf2(e1370) mutants was analyzed using real-time PCR.The reporter GFP::LGG-1 was used to verify whether autophagy was induced in motor neurons.Results:Expression of G93A SOD1 in motor neurons caused age-dependent motor defects accompanied by significant SOD1 aggregation and axon guidance failure.After 12 d,over 80% of the G93A worms became paralyzed,whereas less than 10% of the controls showed a paralytic phenotype.In the daf-2(e1370) mutants of C.elegans,the levels of autophagic genes bec-1,atg-7,Igg-1,and atg-18 were upregulated by approximately 1.5-fold,the level of unc-51 increased by approximately fourfold,and autophagosomes in motor neurons was markedly increased.Crossing the daf-2(e1370) mutation into the G93A SOD1 mutant worms significantly ameliorated the motor defects,SOD1 aggregation,and axon guidance failure.Conclusion:G93A SOD1 expression in motor neurons of C.elegans results in characteristic alterations of ALS.Increased autophagy protects C.elegans motor neurons against the toxicity of mutant SOD1.

  15. Precision autophagy: Will the next wave of selective autophagy markers and specific autophagy inhibitors feed clinical pipelines?

    Science.gov (United States)

    Lebovitz, Chandra B; DeVorkin, Lindsay; Bosc, Damien; Rothe, Katharina; Singh, Jagbir; Bally, Marcel; Jiang, Xiaoyan; Young, Robert N; Lum, Julian J; Gorski, Sharon M

    2015-01-01

    Research presented at the Vancouver Autophagy Symposium (VAS) 2014 suggests that autophagy's influence on health and disease depends on tight regulation and precision targeting of substrates. Discussions recognized a pressing need for robust biomarkers that accurately assess the clinical utility of modulating autophagy in disease contexts. Biomarker discovery could flow from investigations of context-dependent triggers, sensors, and adaptors that tailor the autophagy machinery to achieve target specificity. In his keynote address, Dr. Vojo Deretic (University of New Mexico) described the discovery of a cargo receptor family that utilizes peptide motif-based cargo recognition, a mechanism that may be more precise than generic substrate tagging. The keynote by Dr. Alec Kimmelman (Harvard Medical School) emphasized that unbiased screens for novel selective autophagy factors may accelerate the development of autophagy-based therapies. Using a quantitative proteomics screen for de novo identification of autophagosome substrates in pancreatic cancer, Kimmelman's group discovered a new type of selective autophagy that regulates bioavailable iron. Additional presentations revealed novel autophagy regulators and receptors in metabolic diseases, proteinopathies, and cancer, and outlined the development of specific autophagy inhibitors and treatment regimens that combine autophagy modulation with anticancer therapies. VAS 2014 stimulated interdisciplinary discussions focused on the development of biomarkers, drugs, and preclinical models to facilitate clinical translation of key autophagy discoveries.

  16. Long-term exposure to 835 MHz RF-EMF induces hyperactivity, autophagy and demyelination in the cortical neurons of mice

    Science.gov (United States)

    Kim, Ju Hwan; Yu, Da-Hyeon; Huh, Yang Hoon; Lee, Eun Ho; Kim, Hyung-Gun; Kim, Hak Rim

    2017-01-01

    Radiofrequency electromagnetic field (RF-EMF) is used globally in conjunction with mobile communications. There are public concerns of the perceived deleterious biological consequences of RF-EMF exposure. This study assessed neuronal effects of RF-EMF on the cerebral cortex of the mouse brain as a proxy for cranial exposure during mobile phone use. C57BL/6 mice were exposed to 835 MHz RF-EMF at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day during 12 weeks. The aim was to examine activation of autophagy pathway in the cerebral cortex, a brain region that is located relatively externally. Induction of autophagy genes and production of proteins including LC3B-II and Beclin1 were increased and accumulation of autolysosome was observed in neuronal cell bodies. However, proapoptotic factor Bax was down-regulted in the cerebral cortex. Importantly, we found that RF-EMF exposure led to myelin sheath damage and mice displayed hyperactivity-like behaviour. The data suggest that autophagy may act as a protective pathway for the neuronal cell bodies in the cerebral cortex during radiofrequency exposure. The observations that neuronal cell bodies remained structurally stable but demyelination was induced in cortical neurons following prolonged RF-EMF suggests a potential cause of neurological or neurobehavioural disorders. PMID:28106136

  17. 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.

  18. 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.

  19. Overexpression of FOXO3, MYD88, and GAPDH Identified by Suppression Subtractive Hybridization in Esophageal Cancer Is Associated with Autophagy.

    Science.gov (United States)

    Soltany-Rezaee-Rad, Mohammad; Mottaghi-Dastjerdi, Negar; Setayesh, Neda; Roshandel, Gholamreza; Ebrahimifard, Farzaneh; Sepehrizadeh, Zargham

    2014-01-01

    To find genes involved in tumorigenesis and the development of esophageal cancer, the suppression subtractive hybridization (SSH) method was used to identify genes that are overexpressed in esophageal cancer tissues compared to normal esophageal tissues. In our SSH library, the forkhead box O3 (FOXO3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and myeloid differentiation primary response 88 (MYD88) genes were the most highly upregulated genes, and they were selected for further studies because of their potential role in the induction of autophagy. Upregulation of these genes was also observed in clinical samples using qRT-PCR. In addition, coexpression analysis of the autophagy-related genes Beclin1, ATG12, Gabarapl, PIK3C3, and LC3 demonstrated a significant correlation between the differentially overexpressed genes and autophagy. Autophagy is an important mechanism in tumorigenesis and the development of chemoresistance in cancer cells. The upregulation of FOXO3, GAPDH, and MYD88 variants in esophageal cancer suggests a role for autophagy and provides new insight into the biology of esophageal cancer. We propose that FOXO3, GAPDH, and MYD88 are novel targets for combating autophagy in esophageal cancer.

  20. Overexpression of FOXO3, MYD88, and GAPDH Identified by Suppression Subtractive Hybridization in Esophageal Cancer Is Associated with Autophagy

    Directory of Open Access Journals (Sweden)

    Mohammad Soltany-Rezaee-Rad

    2014-01-01

    Full Text Available To find genes involved in tumorigenesis and the development of esophageal cancer, the suppression subtractive hybridization (SSH method was used to identify genes that are overexpressed in esophageal cancer tissues compared to normal esophageal tissues. In our SSH library, the forkhead box O3 (FOXO3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, and myeloid differentiation primary response 88 (MYD88 genes were the most highly upregulated genes, and they were selected for further studies because of their potential role in the induction of autophagy. Upregulation of these genes was also observed in clinical samples using qRT-PCR. In addition, coexpression analysis of the autophagy-related genes Beclin1, ATG12, Gabarapl, PIK3C3, and LC3 demonstrated a significant correlation between the differentially overexpressed genes and autophagy. Autophagy is an important mechanism in tumorigenesis and the development of chemoresistance in cancer cells. The upregulation of FOXO3, GAPDH, and MYD88 variants in esophageal cancer suggests a role for autophagy and provides new insight into the biology of esophageal cancer. We propose that FOXO3, GAPDH, and MYD88 are novel targets for combating autophagy in esophageal cancer.

  1. Autophagy and obesity%自噬和肥胖

    Institute of Scientific and Technical Information of China (English)

    张林; 胡茂清

    2015-01-01

    [Summary] Autophagy is an intracellular degradation process by which the damaged organelles and macromolecules are lysosomal dependently degraded by auto‐phagocyte under the control of autophagy‐related genes. The autophagy level in hypothalamus and adipose tissue changes in obese individuals.Autophagy participates in the regulation of food intake and energy balance ,and associates with adipocyte differentiation and adipogenesis. More and more attention has been paid to the relationship between autophagy and obesity w hich may guide the new research direction of pathogenesis study and therapy of obesity.%自噬是在相关基因的调控下,自噬细胞溶酶体依赖性的降解细胞内受损的细胞器及大分子物质的过程。肥胖个体下丘脑、脂肪组织自噬水平改变,自噬参与了下丘脑控制进食及能量平衡调节,并且与脂肪细胞的分化、脂肪形成有关。自噬与肥胖发生的关系日益受到重视,为肥胖的发病机制研究及治疗提供了新的方向。

  2. 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.

  3. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

    Science.gov (United States)

    Wise, Andrew K; Tu, Tian; Atkinson, Patrick J; Flynn, Brianna O; Sgro, Beatrice E; Hume, Cliff; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2011-08-01

    A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the

  4. Anti-tumor immunity, autophagy and chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Gy(o)rgyi Müzes; Ferenc Sipos

    2012-01-01

    Autophagy or self-digestion of cells is activated upon various stressful stimuli and has been found to be a survival and drug resistance pathway in cancer.However,genetic studies support that autophagy can act as a tumor suppressor.Furthermore,defective autophagy is implicated in tumorigenesis,as well.The precise impact of autophagy on malignant transformation has not yet been clarified,but recent data suggest that this complex process is mainly directed by cell types,phases,genetic background and microenvironment.Relation of autophagy to anticancer immune responses may indicate a novel aspect in cancer chemotherapy.

  5. The cellular decision between apoptosis and autophagy

    Institute of Scientific and Technical Information of China (English)

    Yong-Jun Fan; Wei-Xing Zong

    2013-01-01

    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.

  6. AUTOPHAGY AND IL-1 FAMILY CYTOKINES

    Directory of Open Access Journals (Sweden)

    James Harris

    2013-01-01

    Full Text Available Autophagy is an important intracellular homeostatic mechanism for the targeting of cytosolic constituents, including organelles, for lysosomal degradation. Autophagy plays roles in numerous physiological processes, include immune cell responses to endogenous and exogenous pathogenic stimuli. Moreover, autophagy has a potentially pivotal role in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. This review focuses specifically on the role autophagy plays in regulating the production, processing and secretion of IL-1 family cytokines.

  7. MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy.

    Science.gov (United States)

    Paillas, Salome; Causse, Annick; Marzi, Laetitia; de Medina, Philippe; Poirot, Marc; Denis, Vincent; Vezzio-Vie, Nadia; Espert, Lucile; Arzouk, Hayat; Coquelle, Arnaud; Martineau, Pierre; Del Rio, Maguy; Pattingre, Sophie; Gongora, Céline

    2012-07-01

    Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.

  8. Autophagy positively regulates the CD44(+) CD24(-/low) breast cancer stem-like phenotype.

    Science.gov (United States)

    Cufí, Sílvia; Vazquez-Martin, Alejandro; Oliveras-Ferraros, Cristina; Martin-Castillo, Begoña; Vellon, Luciano; Menendez, Javier A

    2011-11-15

    The molecular mechanisms used by breast cancer stem cells (BCSCs) to survive and/or maintain their undifferentiated CD44(+) CD24(-/low ) mesenchymal-like antigenic state remains largely unexplored. Autophagy, a key homeostatic process of cytoplasmic degradation and recycling evolved to respond to stress conditions, might be causally fundamental in the biology of BCSCs. Stable & specific knockdown of autophagy-regulatory genes by lentiviral-delivered small hairpin (sh) RNA drastically decreased the number of JIMT-1 epithelial BC cells bearing CD44(+) CD24(-/low) cell-surface antigens from ~75% in parental and control (-) shRNA-transduced cells to 26% and 7% in ATG8/LC3 shRNA- and ATG12 shRNA-transduced cells, respectively. Autophagy inhibition notably enhanced transcriptional activation of CD24 gene, potentiating the epithelial-like phenotype of CD44(+) CD24(+) cells versus the mesenchymal CD44(+) CD24(-/low ) progeny. EMT-focused Real Time RT-PCR profiling revealed that genetic ablation of autophagy transcriptionally repressed the gene coding for the mesenchymal filament vimentin (VIM). shRNA-driven silencing of the ATG12 gene and disabling the final step in the autophagy pathway by the antimalarial drug chloroquine both prevented TGFb1-induced accumulation of vimentin in JIMT-1 cells. Knockdown of autophagy-specific genes was sufficient also to increase by up to 11-times the number of CD24(+) cells in MDA-MB-231 cells, a BC model of mesenchymal origin that is virtually composed of CD44(+) CD24(-/low ) cells. Chloroquine treatment augmented the number of CD24(+) cells and concomitantly reduced constitutive overexpression of vimentin in MDA-MB-231 cells. This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of BCSCs.

  9. NAC1 and HMGB1 enter a partnership for manipulating autophagy.

    Science.gov (United States)

    Zhang, Yi; Yang, Jay W; Ren, Xingcong; Yang, Jin-Ming

    2011-12-01

    Our recent study revealed a new role of nucleus accumbens-1 (NAC1), a transcription factor belonging to the BTB/POZ gene family, in regulating autophagy. Moreover, we found that the high-mobility group box 1 (HMGB1), a chromatin-associated nuclear protein acting as an extracellular damage associated molecular pattern molecule (DAMP), is the downstream executor of NAC1 in modulating autophagy. In response to stress such as therapeutic insults, NAC1 increases the expression, cytosolic translocation and release of HMGB1; elevated level of the cytoplasmic HMGB1 leads to activation of autophagy. The NAC1-HMGB1 partnership may represent a previously unrecognized pathway that regulates autophagy in response to various stresses such as chemotherapy.

  10. 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

  11. The symphony of autophagy and calcium signaling.

    Science.gov (United States)

    Yao, Zhiyuan; Klionsky, Daniel J

    2015-01-01

    Posttranslational regulation of macroautophagy (hereafter autophagy), including phosphorylating and dephosphorylating components of the autophagy-related (Atg) core machinery and the corresponding upstream transcriptional factors, is important for the precise modulation of autophagy levels. Several kinases that are involved in phosphorylating autophagy-related proteins have been identified in both yeast and mammalian cells. However, there has been much less research published with regard to the identification of the complementary phosphatases that function in autophagy. A recent study identified PPP3/calcineurin, a calcium-dependent phosphatase, as a regulator of autophagy, and demonstrated that one of the key targets of PPP3/calcineurin is TFEB, a master transcriptional factor that controls autophagy and lysosomal function in mammalian cells.

  12. 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.

  13. Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

    Institute of Scientific and Technical Information of China (English)

    Jiang-Li DONG; Bo ZHOU; Gang SHENG; Tao WANG

    2005-01-01

    Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized (sVP6). The VP6 and sVp6genes were transformed into tobacco (Nicotiana tabacum L.) plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein (TSP). Then, BALB/c female mice that had been gavaged weekly with 10 mg TSP containing 34 μg VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.

  14. 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.

  15. Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation.

    Science.gov (United States)

    Rialdi, Alex; Campisi, Laura; Zhao, Nan; Lagda, Arvin Cesar; Pietzsch, Colette; Ho, Jessica Sook Yuin; Martinez-Gil, Luis; Fenouil, Romain; Chen, Xiaoting; Edwards, Megan; Metreveli, Giorgi; Jordan, Stefan; Peralta, Zuleyma; Munoz-Fontela, Cesar; Bouvier, Nicole; Merad, Miriam; Jin, Jian; Weirauch, Matthew; Heinz, Sven; Benner, Chris; van Bakel, Harm; Basler, Christopher; García-Sastre, Adolfo; Bukreyev, Alexander; Marazzi, Ivan

    2016-05-27

    The host innate immune response is the first line of defense against pathogens and is orchestrated by the concerted expression of genes induced by microbial stimuli. Deregulated expression of these genes is linked to the initiation and progression of diseases associated with exacerbated inflammation. We identified topoisomerase 1 (Top1) as a positive regulator of RNA polymerase II transcriptional activity at pathogen-induced genes. Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses as well as bacterial products. Therapeutic pharmacological inhibition of Top1 protected mice from death in experimental models of lethal inflammation. Our results indicate that Top1 inhibition could be used as therapy against life-threatening infections characterized by an acutely exacerbated immune response.

  16. Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiaobo Chen; Yongxiang Li; Weiping Dong; Yang Jiao; Jianming Tan

    2007-01-01

    Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively.The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index (SI) was calculated. Glucose-stimulated insulin release was usedto assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index(SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets(P < 0.05).Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

  17. Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition.

    Science.gov (United States)

    Xu, Kang; Chen, Weijian; Wang, Xiaofei; Peng, Yan; Liang, Anjing; Huang, Dongsheng; Li, Chunhai; Ye, Wei

    2015-09-01

    Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and intervertebral disc degeneration. However, the autophagy induced by TNF‑α and IL‑1β and the corresponding molecular mechanism appear to be cell‑type dependent. The effect and mechanism of autophagy regulated by TNF‑α and IL‑1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF‑α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy‑related gene expression in rat NP cells following TNF‑α and IL‑1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF‑κB), c‑Jun N‑terminal kinase (JNK), extracellular signal‑regulated kinases and p38 mitogen‑activated protein kinase inhibitors and TNF‑α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF‑κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF‑κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions.

  18. FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation.

    Science.gov (United States)

    Dunlop, Elaine A; Seifan, Sara; Claessens, Tijs; Behrends, Christian; Kamps, Miriam Af; Rozycka, Ewelina; Kemp, Alain J; Nookala, Ravi K; Blenis, John; Coull, Barry J; Murray, James T; van Steensel, Maurice Am; Wilkinson, Simon; Tee, Andrew R

    2014-10-01

    Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

  19. Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium.

    Science.gov (United States)

    Yao, Jingyu; Jia, Lin; Khan, Naheed; Lin, Chengmao; Mitter, Sayak K; Boulton, Michael E; Dunaief, Joshua L; Klionsky, Daniel J; Guan, Jun-Lin; Thompson, Debra A; Zacks, David N

    2015-01-01

    Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1(flox/flox)). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.

  20. 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

  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. Skeletal Muscle-derived Myonectin Activates the Mammalian Target of Rapamycin (mTOR) Pathway to Suppress Autophagy in Liver*

    Science.gov (United States)

    Seldin, Marcus M.; Lei, Xia; Tan, Stefanie Y.; Stanson, Kevin P.; Wei, Zhikui; Wong, G. William

    2013-01-01

    Cells turn on autophagy, an intracellular recycling pathway, when deprived of nutrients. How autophagy is regulated by hormonal signals in response to major changes in metabolic state is not well understood. Here, we provide evidence that myonectin (CTRP15), a skeletal muscle-derived myokine, is a novel regulator of cellular autophagy. Starvation activated liver autophagy, whereas nutrient supplementation following food deprivation suppressed it; the former and latter correlated with reduced and increased expression and circulating levels of myonectin, respectively, suggestive of a causal link. Indeed, recombinant myonectin administration suppressed starvation-induced autophagy in mouse liver and cultured hepatocytes, as indicated by the inhibition of LC3-dependent autophagosome formation, p62 degradation, and expression of critical autophagy-related genes. Reduction in protein degradation is mediated by the PI3K/Akt/mTOR signaling pathway; inhibition of this pathway abrogated the ability of myonectin to suppress autophagy in cultured hepatocytes. Together, our results reveal a novel skeletal muscle-liver axis controlling cellular autophagy, underscoring the importance of hormone-mediated tissue cross-talk in maintaining energy homeostasis. PMID:24187137

  3. Protective gene expression changes elicited by an inherited defect in photoreceptor structure.

    Directory of Open Access Journals (Sweden)

    Yagya V Sharma

    Full Text Available Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE, and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.

  4. 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.

  5. Global Gene Expression Analysis of Cross-Protected Phenotype of Pectobacterium atrosepticum.

    Science.gov (United States)

    Gorshkov, Vladimir; Kwenda, Stanford; Petrova, Olga; Osipova, Elena; Gogolev, Yuri; Moleleki, Lucy N

    2017-01-01

    The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features

  6. Global Gene Expression Analysis of Cross-Protected Phenotype of Pectobacterium atrosepticum

    Science.gov (United States)

    Gorshkov, Vladimir; Kwenda, Stanford; Petrova, Olga; Osipova, Elena; Gogolev, Yuri; Moleleki, Lucy N.

    2017-01-01

    The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features

  7. HSF-1 is involved in attenuating the release of inflammatory cytokines induced by LPS through regulating autophagy.

    Science.gov (United States)

    Tong, Zhongyi; Jiang, Bimei; Zhang, Lingli; Liu, Yanjuan; Gao, Min; Jiang, Yu; Li, Yuanbin; Lu, Qinglan; Yao, Yongming; Xiao, Xianzhong

    2014-05-01

    Autophagy plays a protective role in endotoxemic mice. Heat shock factor 1 (HSF-1) also plays a crucial protective role in endotoxemic mice by decreasing inflammatory cytokines. The purpose of this study was to determine whether HSF-1 is involved in attenuating the release of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated mice and peritoneal macrophages (PMs) through regulating autophagy activity. Autophagosome formation in HSF-1(+/+) and HSF-1(-/-) mice and PMs stimulated by LPS was examined by Western blotting and immunofluorescence. Lipopolysaccharide-induced autophagy and inflammatory cytokines were examined in HSF-1(+/+) and HSF-1(-/-) PMs treated with 3-methyladenine (3-MA) or rapamycin. Results showed that LPS-induced autophagy was elevated transiently at 12 h but declined at 24 h in the livers and lungs of mice. Higher levels of inflammatory cytokines and lower autophagy activity were detected in HSF-1(-/-) mice and PMs compared with HSF-1(+/+) mice and PMs. Interestingly, LPS-induced release of inflammatory cytokines did not further increase in HSF-1(-/-) PMs treated with 3-MA but aggravated in HSF-1(+/+) PMs. Lipopolysaccharide-induced autophagy did not decrease in HSF-1(-/-) PMs treated with 3-MA but decreased in HSF-1 PMs(+/+). Taken together, our results suggested that HSF-1 attenuated the release of inflammatory cytokines induced by LPS by regulating autophagy activity.

  8. Protective Effect of CRHR1 Gene Variants on the Development of Adult Depression Following Childhood Maltreatment

    Science.gov (United States)

    Polanczyk, Guilherme; Caspi, Avshalom; Williams, Benjamin; Price, Thomas S.; Danese, Andrea; Sugden, Karen; Uher, Rudolf; Poulton, Richie; Moffitt, Terrie E.

    2013-01-01

    Context A previous study reported a gene × environment interaction in which a haplotype in the corticotropin-releasing hormone receptor 1 gene (CRHR1) was associated with protection against adult depressive symptoms in individuals who were maltreated as children (as assessed by the Childhood Trauma Questionnaire [CTQ]). Objective To replicate the interaction between childhood maltreatment and a TAT haplotype formed by rs7209436, rs110402, and rs242924 in CRHR1, predicting adult depression. Design Two prospective longitudinal cohort studies. Setting England and New Zealand. Participants Participants in the first sample were women in the E-Risk Study (N= 1116), followed up to age 40 years with 96% retention. Participants in the second sample were men and women in the Dunedin Study (N= 1037), followed up to age 32 years with 96% retention. Main Outcome Measure Research diagnoses of pastyear and recurrent major depressive disorder. Results In the E-Risk Study, the TAT haplotype was associated with a significant protective effect. In this effect, women who reported childhood maltreatment on the CTQ were protected against depression. In the Dunedin Study, which used a different type of measure of maltreatment, this finding was not replicated. Conclusions A haplotype in CRHR1 has been suggested to exert a protective effect against adult depression among research participants who reported maltreatment on the CTQ, a measure that elicits emotional memories. This suggests the hypothesis that CRHR1’s protective effect may relate to its function in the consolidation of memories of emotionally arousing experiences. PMID:19736354

  9. BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells

    Science.gov (United States)

    Goulielmaki, Maria; Koustas, Evangelos; Moysidou, Eirini; Vlassi, Margarita; Sasazuki, Takehiko; Shirasawa, Senji; Zografos, George; Oikonomou, Eftychia; Pintzas, Alexander

    2016-01-01

    Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer – both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene. PMID:26802026

  10. BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells.

    Science.gov (United States)

    Goulielmaki, Maria; Koustas, Evangelos; Moysidou, Eirini; Vlassi, Margarita; Sasazuki, Takehiko; Shirasawa, Senji; Zografos, George; Oikonomou, Eftychia; Pintzas, Alexander

    2016-02-23

    Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.

  11. Posttranslational modification of autophagy-related proteins in macroautophagy.

    Science.gov (United States)

    Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J; Tang, Daolin

    2015-01-01

    Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.

  12. 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.

  13. Age-regulated function of autophagy in the mouse inner ear.

    Science.gov (United States)

    de Iriarte Rodríguez, Rocío; Pulido, Sara; Rodríguez-de la Rosa, Lourdes; Magariños, Marta; Varela-Nieto, Isabel

    2015-12-01

    Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles during early development in the chicken embryo and in the response to otic injury in the adult mouse. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation. We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the

  14. 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

  15. The critical role of quercetin in autophagy and apoptosis in HeLa cells.

    Science.gov (United States)

    Wang, Yijun; Zhang, Wei; Lv, Qiongying; Zhang, Juan; Zhu, Dingjun

    2016-01-01

    In recent years, the effects of quercetin on autophagy and apoptosis of cancer cells have been widely reported, while effects on HeLa cells are still unclear. Here, HeLa cells were subjected to quercetin treatment, and then proliferation, apoptosis, and autophagy were evaluated using MTT, flow cytometry, and MDC staining, respectively. The LC3-I/II, Beclin 1, active caspase-3, and S6K1 phosphorylation were detected using Western blot assay. The ultrastructure of HeLa was observed via transmission electron microscope (TEM). Our findings showed that quercetin can dose-dependently inhibit the growth of HeLa cells. The MDC fluorescence was enhanced with increased concentration of quercetin and hit a plateau at 50 μmol/l. Western blot assay revealed that LC3-I/II ratio, Beclin 1, and active caspase-3 protein were enforced in a dose-dependent method. However, the phosphorylation of S6K1 gradually decreased, concomitant with an increase of autophagy. In addition, TEM revealed that the number of autophagic vacuoles was peaked at 50 μmol/l of quercetin. Besides, interference of autophagy with 3-MA led to proliferation inhibition and increased apoptosis in HeLa cells, accompanied by the decreased LC3-I/II conversion and the increased active caspase-3. In conclusion, quercetin can inhibit HeLa cell proliferation and induce protective autophagy at low concentrations; thus, 3-MA plus quercetin would suppress autophagy and effectively increased apoptosis.

  16. Ordered bulk degradation via autophagy

    DEFF Research Database (Denmark)

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

    2008-01-01

    at 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...

  17. Sucrose induces vesicle accumulation and autophagy.

    Science.gov (United States)

    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes.

  18. HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression

    Directory of Open Access Journals (Sweden)

    Meiri Eti

    2009-02-01

    Full Text Available Abstract Background RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression. Results Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well. Conclusion The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

  19. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

    Science.gov (United States)

    Luan, Qi; Jin, Lei; Jiang, Chen Chen; Tay, Kwang Hong; Lai, Fritz; Liu, Xiao Ying; Liu, Yi Lun; Guo, Su Tang; Li, Chun Ying; Yan, Xu Guang; Tseng, Hsin-Yi; Zhang, Xu Dong

    2015-01-01

    Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

  20. 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.

  1. Autophagy-preferential degradation of MIR224 participates in hepatocellular carcinoma tumorigenesis.

    Science.gov (United States)

    Lan, Sheng-Hui; Wu, Shan-Ying; Zuchini, Roberto; Lin, Xi-Zhang; Su, Ih-Jen; Tsai, Ting-Fen; Lin, Yen-Ju; Wu, Cheng-Tao; Liu, Hsiao-Sheng

    2014-09-01

    Autophagy and microRNA (miRNA) are important regulators during cancer cell tumorigenesis. Impaired autophagy and high expression of the oncogenic microRNA MIR224 are prevalent in hepatocellular carcinoma (HCC); however, the relationship between the 2 phenomena remains elusive. In this study, we are the first to reveal that autophagy selectively regulates MIR224 expression through an autophagosome-mediated degradation system. Based on this finding, we further demonstrated that in hepatitis B virus (HBV)-related HCC, aberrant autophagy (low autophagic activity) results in accumulation of MIR224 and decreased expression of the target gene Smad4, which leads to increased cell migration and tumor formation. Preferential recruitment of MIR224 into the autophagosome was clearly demonstrated by a) miRNA in situ hybridization under confocal microscopy, and b) immunogold labeling of MIR224 under electron microscopy compared with a ubiquitously expressed microRNA MIRlet7e/let-7. Furthermore, we found that off-label use of amiodarone, an antiarrhythmic agent, effectively suppressed HCC tumorigenesis through autophagy-mediated MIR224 degradation both in vitro and in vivo. In summary, we identified amiodarone as a new autophagy inducer, which may provide an alternative approach in HCC therapy through a novel tumor suppression mechanism.

  2. Blocking tumor growth by targeting autophagy and SQSTM1 in vivo.

    Science.gov (United States)

    Wei, Huijun; Guan, Jun-Lin

    2015-01-01

    Autophagy is a highly conserved cellular process for degradation of bulk cytoplasmic materials in response to starvation and maintenance of cellular homeostasis. Dysfunction of autophagy is implicated in a variety of diseases including cancer. In a recent study, we devised a system for inducible deletion of an essential autophagy gene Rb1cc1/Fip200 in established tumor cells in vivo and showed that Rb1cc1 is required for maintaining tumor growth. We further investigated the role of the accumulated SQSTM1 in Rb1cc1-null autophagy-deficient tumor cells. To our surprise, the increased SQSTM1 was not responsible for the inhibition of tumor growth, but rather supported the residual growth of tumors (i.e., partially compensated for the defective growth caused by Rb1cc1 deletion). Further analysis indicated that SQSTM1 promoted tumor growth in autophagy-deficient cells at least partially through its activation of the NFKB signaling pathway. A working model is proposed to account for our findings, which suggest that targeting both autophagy and the consequently increased SQSTM1 may be exploited for developing more effective cancer therapies.

  3. Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium.

    Science.gov (United States)

    Chen, Zhi-Hua; Wu, Yin-Fang; Wang, Ping-Li; Wu, Yan-Ping; Li, Zhou-Yang; Zhao, Yun; Zhou, Jie-Sen; Zhu, Chen; Cao, Chao; Mao, Yuan-Yuan; Xu, Feng; Wang, Bei-Bei; Cormier, Stephania A; Ying, Song-Min; Li, Wen; Shen, Hua-Hao

    2016-01-01

    Environmental ultrafine particulate matter (PM) is capable of inducing airway injury, while the detailed molecular mechanisms remain largely unclear. Here, we demonstrate pivotal roles of autophagy in regulation of inflammation and mucus hyperproduction induced by PM containing environmentally persistent free radicals in human bronchial epithelial (HBE) cells and in mouse airways. PM was endocytosed by HBE cells and simultaneously triggered autophagosomes, which then engulfed the invading particles to form amphisomes and subsequent autolysosomes. Genetic blockage of autophagy markedly reduced PM-induced expression of inflammatory cytokines, e.g. IL8 and IL6, and MUC5AC in HBE cells. Mice with impaired autophagy due to knockdown of autophagy-related gene Becn1 or Lc3b displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Interference of the autophagic flux by lysosomal inhibition resulted in accumulated autophagosomes/amphisomes, and intriguingly, this process significantly aggravated the IL8 production through NFKB1, and markedly attenuated MUC5AC expression via activator protein 1. These data indicate that autophagy is required for PM-induced airway epithelial injury, and that inhibition of autophagy exerts therapeutic benefits for PM-induced airway inflammation and mucus hyperproduction, although they are differentially orchestrated by the autophagic flux.

  4. Autophagy is induced in the skeletal muscle of cachectic cancer patients

    Science.gov (United States)

    Aversa, Zaira; Pin, Fabrizio; Lucia, Simone; Penna, Fabio; Verzaro, Roberto; Fazi, Maurizio; Colasante, Giuseppina; Tirone, Andrea; Fanelli, Filippo Rossi; Ramaccini, Cesarina; Costelli, Paola; Muscaritoli, Maurizio

    2016-01-01

    Basal rates of autophagy can be markedly accelerated by environmental stresses. Recently, autophagy has been involved in cancer-induced muscle wasting. Aim of this study has been to evaluate if autophagy is induced in the skeletal muscle of cancer patients. The expression (mRNA and protein) of autophagic markers has been evaluated in intraoperative muscle biopsies. Beclin-1 protein levels were increased in cachectic cancer patients, suggesting autophagy induction. LC3B-I protein levels were not significantly modified. LC3B-II protein levels were significantly increased in cachectic cancer patients suggesting either increased autophagosome formation or reduced autophagosome turnover. Conversely, p62 protein levels were increased in cachectic and non-cachectic cancer patients, suggesting impaired autophagosome clearance. As for mitophagy, both Bnip3 and Nix/Bnip3L show a trend to increase in cachectic patients. In the same patients, Parkin levels significantly increased, while PINK1 was unchanged. At gene level, Beclin-1, p-62, BNIP3, NIX/BNIP3L and TFEB mRNAs were not significantly modulated, while LC3B and PINK1 mRNA levels were increased and decreased, respectively, in cachectic cancer patients. Autophagy is induced in the skeletal muscle of cachectic cancer patients, although autophagosome clearance appears to be impaired. Further studies should evaluate whether modulation of autophagy could represent a relevant therapeutic strategy in cancer cachexia. PMID:27459917

  5. 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

    2016-03-08

    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.

  6. Activation of autophagy in response to nanosecond pulsed electric field exposure.

    Science.gov (United States)

    Ullery, Jody C; Tarango, Melissa; Roth, Caleb C; Ibey, Bennett L

    2015-03-06

    Previous work demonstrated significant changes in cellular membranes following exposure of cells to nanosecond pulsed electric fields (nsPEF), including nanoporation and increases in intracellular calcium concentration. While it is known that nsPEF exposure can cause cell death, how cells repair and survive nsPEF-induced cellular damage is not well understood. In this paper, we investigated whether autophagy is stimulated following nsPEF exposure to repair damaged membranes, proteins, and/or organelles in a pro-survival response. We hypothesized that autophagy is activated to repair nsPEF-induced plasma membrane damage and overwhelming this compensatory mechanism results in cell death. Activation of autophagy and subsequent cell death pathways were assessed measuring toxicity, gene and protein expression of autophagy markers, and by monitoring autophagosome formation and maturation using fluorescent microscopy. Results show that autophagy is activated at subtoxic nsPEF doses, as a compensatory mechanism to repair membrane damage. However, prolonged exposure results in increased cell death and a concomitant decrease in autophagic markers. These results suggest that cells take an active role in membrane repair, through autophagy, following exposure to nsPEF.

  7. Hormetic heat stress and HSF-1 induce autophagy to improve survival and proteostasis in C. elegans

    Science.gov (United States)

    Kumsta, Caroline; Chang, Jessica T.; Schmalz, Jessica; Hansen, Malene

    2017-01-01

    Stress-response pathways have evolved to maintain cellular homeostasis and to ensure the survival of organisms under changing environmental conditions. Whereas severe stress is detrimental, mild stress can be beneficial for health and survival, known as hormesis. Although the universally conserved heat-shock response regulated by transcription factor HSF-1 has been implicated as an effector mechanism, the role and possible interplay with other cellular processes, such as autophagy, remains poorly understood. Here we show that autophagy is induced in multiple tissues of Caenorhabditis elegans following hormetic heat stress or HSF-1 overexpression. Autophagy-related genes are required for the thermoresistance and longevity of animals exposed to hormetic heat shock or HSF-1 overexpression. Hormetic heat shock also reduces the progressive accumulation of PolyQ aggregates in an autophagy-dependent manner. These findings demonstrate that autophagy contributes to stress resistance and hormesis, and reveal a requirement for autophagy in HSF-1-regulated functions in the heat-shock response, proteostasis and ageing. PMID:28198373

  8. Development of potent autophagy inhibitors that sensitize oncogenic BRAF V600E mutant melanoma tumor cells to vemurafenib.

    Science.gov (United States)

    Goodall, Megan L; Wang, Tong; Martin, Katie R; Kortus, Matthew G; Kauffman, Audra L; Trent, Jeffrey M; Gately, Stephen; MacKeigan, Jeffrey P

    2014-06-01

    Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.

  9. Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages.

    Science.gov (United States)

    Rekha, Rokeya Sultana; Rao Muvva, S S V Jagadeeswara; Wan, Min; Raqib, Rubhana; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H; Agerberth, Birgitta

    2015-01-01

    LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca(2+), and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.

  10. Autophagy and IL-1 family cytokines

    Directory of Open Access Journals (Sweden)

    James eHarris

    2013-04-01

    Full Text Available Autophagy is an important intracellular homeostatic mechanism for the targeting of cytosolic constituents, including organelles, for lysosomal degradation. Autophagy plays roles in numerous physiological processes, including immune cell responses to endogenous and exogenous pathogenic stimuli. Moreover, autophagy has a potentially pivotal role to play in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. As a result, autophagy acts a key modulator of IL-1β and IL-18, as well as IL-1α, release. This review focuses specifically on the role autophagy plays in regulating the production, processing and secretion of IL-1 and IL-18 and the consequences of this important function.

  11. Autophagy and IL-1 Family Cytokines.

    Science.gov (United States)

    Harris, James

    2013-01-01

    Autophagy is an important intracellular homeostatic mechanism for the targeting of cytosolic constituents, including organelles, for lysosomal degradation. Autophagy plays roles in numerous physiological processes, including immune cell responses to endogenous and exogenous pathogenic stimuli. Moreover, autophagy has a potentially pivotal role to play in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. As a result, autophagy acts a key modulator of IL-1β and IL-18, as well as IL-1α, release. This review focuses specifically on the role autophagy plays in regulating the production, processing, and secretion of IL-1 and IL-18 and the consequences of this important function.

  12. Autophagy gets in on the regulatory act

    Institute of Scientific and Technical Information of China (English)

    Steven K. Backues; Daniel J. Klionsky

    2011-01-01

    Autophagy down-regulates the Wnt signal transduction pathway via targeted degradation of a key signaling protein. This may provide an explanation for autophagy's role in tumor suppression.%@@ The eukaryotic cell has at its disposal two primary methods for getting rid of unwanted proteins: the proteasome and autophagy.The proteasome is a large protein complex comprising regulatory and proteolytic subunits whose core function is the degradation of damaged or misfolded proteins.

  13. Autophagy and apoptosis: where do they meet?

    Science.gov (United States)

    Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Durgesh Nandini; Bhutia, Sujit Kumar

    2014-04-01

    Autophagy and apoptosis are two important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Recent advances have elucidated the key players and their molecular circuitry. For instance, the discovery of Beclin-1's interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis, which functions by interacting with both Beclin-1 and Bax/Bak respectively. When localized to the endoplasmic reticulum and mitochondria, Bcl-2 inhibits autophagy. Cellular stress causes the displacement of Bcl-2 from Beclin-1 and Bax, thereby triggering autophagy and apoptosis, respectively. The induction of autophagy or apoptosis results in disruption of complexes by BH3-only proteins and through post-translational modification. The mechanisms linking autophagy and apoptosis are not fully defined; however, recent discoveries have revealed that several apoptotic proteins (e.g., PUMA, Noxa, Nix, Bax, XIAP, and Bim) modulate autophagy. Moreover, autophagic proteins that control nucleation and elongation regulate intrinsic apoptosis through calpain- and caspase-mediated cleavage of autophagy-related proteins, which switches the cellular program from autophagy to apoptosis. Similarly, several autophagic proteins are implicated in extrinsic apoptosis. This highlights a dual cellular role for autophagy. On one hand, autophagy degrades damaged mitochondria and caspases, and on the other hand, it provides a membrane-based intracellular platform for caspase processing in the regulation of apoptosis. In this review, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis and describe the mechanisms controlling cell survival and cell death.

  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. The dynamic nature of autophagy in cancer.

    Science.gov (United States)

    Kimmelman, Alec C

    2011-10-01

    Macroautophagy (referred to hereafter as autophagy) is a highly regulated cellular process that serves to remove damaged proteins and organelles from the cell. Autophagy contributes to an array of normal and pathological processes, and has recently emerged as a key regulator of multiple aspects of cancer biology. The role of autophagy in cancer is complex and is likely dependent on tumor type, stage, and genetic context. This complexity is illustrated by the identification of settings where autophagy acts potently to either promote or inhibit tumorigenesis. In this review, I discuss the underlying basis for these opposing functions and propose a model suggesting a dynamic role for autophagy in malignancy. Collectively, the data point to autophagy as serving as a barrier to limit tumor initiation. Once neoplastic lesions are established, it appears that adaptive changes occur that now result in positive roles for autophagy in malignant progression and in subsequent tumor maintenance. Remarkably, constitutive activation of autophagy is critical for continued growth of some tumors, serving to both reduce oxidative stress and provide key intermediates to sustain cell metabolism. Autophagy is also induced in response to cancer therapies where it can function as a survival mechanism that limits drug efficacy. These findings have inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. It is now apparent that aberrant control of autophagy is among the key hallmarks of cancer. While much needs to be learned about the regulation and context-dependent biological functions of autophagy, it seems clear that modulation of this process will be an attractive avenue for future cancer therapeutic approaches.

  16. Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.

    Science.gov (United States)

    Kim, Na-Yeon; Han, Byeal-I; Lee, Michael

    2016-01-01

    Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5.

  17. 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.

  18. Icariin Attenuates OGD/R-Induced Autophagy via Bcl-2-Dependent Cross Talk between Apoptosis and Autophagy in PC12 Cells

    Science.gov (United States)

    2016-01-01

    Icariin (ICA), an active component of Epimedium brevicornum Maxim, exerts a variety of neuroprotective effects such as antiapoptosis. However, the mechanisms underlying antiapoptosis of ICA in neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) are unclear. The B-cell lymphoma-2 (Bcl-2) protein family plays an important role in the regulation of apoptosis and autophagy through Bcl-2-dependent cross talk. Bcl-2 suppresses apoptosis by binding to Bax and inhibits autophagy by binding to Beclin-1 which is an autophagy related protein. In the present study, MTT result showed that ICA increased cell viability significantly in OGD/R treated PC12 cells (P < 0.01). Results of western blotting analysis showed that ICA increased Bcl-2 expression significantly and decreased expressions of Bax, cleaved Caspase-3, Beclin-1, and LC3-II significantly in OGD/R treated PC12 cells (P < 0.01). These results suggest that ICA protects PC12 cells from OGD/R induced autophagy via Bcl-2-dependent cross talk between apoptosis and autophagy. PMID:27610184

  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. Regulation of cardiomyocyte autophagy by calcium.

    Science.gov (United States)

    Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

    2016-04-15

    Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy.

  1. Autophagy in term normal human placentas.

    Science.gov (United States)

    Signorelli, P; Avagliano, L; Virgili, E; Gagliostro, V; Doi, P; Braidotti, P; Bulfamante, G P; Ghidoni, R; Marconi, A M

    2011-06-01

    Autophagy is an inducible catabolic process that responds to environment and is essential for cell survival during stress, starvation and hypoxia. Its function in the human placenta it is not yet understood. We collected 14 placentas: 7 at vaginal delivery and 7 at elective caesarean section after uneventful term pregnancies. The presence of autophagy was assessed in different placental areas by immunoblotting, immunohistochemistry and electron microscopy. We found that autophagy is significantly higher in placentas obtained from cesarean section than in those from vaginal delivery. Moreover there is a significant inverse relationship between autophagy and umbilical arterial glucose concentration.

  2. Autophagy : Moving Benchside Promises to Patient Bedsides.

    Science.gov (United States)

    Belaid, Amine; Ndiaye, Papa Diogop; Filippakis, Harilaos; Roux, Jérémie; Röttinger, Éric; Graba, Yacine; Brest, Patrick; Hofman, Paul; Mograbi, Baharia

    2015-01-01

    Survival rates of patients with metastatic or recurrent cancers have remained virtually unchanged during the past 30 years. This fact makes the need for new therapeutic options even more urgent. An attractive option would be to target autophagy, an essential quality control process that degrades toxic aggregates, damaged organelles, and signaling proteins, and acts as a tumor suppressor pathway of tumor initiation. Conversely, other fascinating observations suggest that autophagy supports cancer progression, relapse, metastasis, dormancy and resistance to therapy. This review provides an overview of the contradictory roles that autophagy plays in cancer initiation and progression and discusses the promises and challenges of current strategies that target autophagy for cancer therapy.

  3. The Escherichia coli SOS gene dinF protects against oxidative stress and bile salts.

    Directory of Open Access Journals (Sweden)

    Jerónimo Rodríguez-Beltrán

    Full Text Available DNA is constantly damaged by physical and chemical factors, including reactive oxygen species (ROS, such as superoxide radical (O(2(-, hydrogen peroxide (H(2O(2 and hydroxyl radical (•OH. Specific mechanisms to protect and repair DNA lesions produced by ROS have been developed in living beings. In Escherichia coli the SOS system, an inducible response activated to rescue cells from severe DNA damage, is a network that regulates the expression of more than 40 genes in response to this damage, many of them playing important roles in DNA damage tolerance mechanisms. Although the function of most of these genes has been elucidated, the activity of some others, such as dinF, remains unknown. The DinF deduced polypeptide sequence shows a high homology with membrane proteins of the multidrug and toxic compound extrusion (MATE family. We describe here that expression of dinF protects against bile salts, probably by decreasing the effects of ROS, which is consistent with the observed decrease in H(2O(2-killing and protein carbonylation. These results, together with its ability to decrease the level of intracellular ROS, suggests that DinF can detoxify, either direct or indirectly, oxidizing molecules that can damage DNA and proteins from both the bacterial metabolism and the environment. Although the exact mechanism of DinF activity remains to be identified, we describe for the first time a role for dinF.

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

    Energy Technology Data Exchange (ETDEWEB)

    Abreu, Maria M. [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Sealy, Linda, E-mail: Linda.sealy@vanderbilt.edu [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Department of Molecular Physiology and Biophysics, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States)

    2010-11-15

    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.

  5. Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury

    Science.gov (United States)

    Chiang, Chih-Kang; Wang, Ching-Chia; Lu, Tien-Fong; Huang, Kuo-How; Sheu, Meei-Ling; Liu, Shing-Hwa; Hung, Kuan-Yu

    2016-01-01

    Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney. PMID:27665710

  6. Rapamycin upregulates autophagy by inhibiting the mTOR-ULK1 pathway, resulting in reduced podocyte injury.

    Directory of Open Access Journals (Sweden)

    Lingling Wu

    Full Text Available The podocyte functions as a glomerular filtration barrier. Autophagy of postmitotic cells is an important protective mechanism that is essential for maintaining the homeostasis of podocytes. Exploring an in vivo rat model of passive Heymann nephritis and an in vitro model of puromycin amino nucleotide (PAN-cultured podocytes, we examined the specific mechanisms underlying changing autophagy levels and podocyte injury. In the passive Heymann nephritis model rats, the mammalian target-of-rapamycin (mTOR levels were upregulated in injured podocytes while autophagy was inhibited. In PAN-treated podocytes, mTOR lowered the level of autophagy through the mTOR-ULK1 pathway resulting in damaged podocytes. Rapamycin treatment of these cells reduced podocyte injury by raising the levels of autophagy. These in vivo and in vitro experiments demonstrate that podocyte injury is associated with changes in autophagy levels, and that rapamycin can reduce podocyte injury by increasing autophagy levels via inhibition of the mTOR-ULK1 pathway. These results provide an important theoretical basis for future treatment of diseases involving podocyte injury.

  7. Protection of germline gene expression by the C. elegans Argonaute CSR-1.

    Science.gov (United States)

    Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

    2013-12-23

    In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

  8. Effect of ARHI transfection on apoptosis and autophagy related gene expression profile of PANC1 cells%ARHI基因转染PANC1细胞后对细胞凋亡和自噬相关基因表达谱的影响

    Institute of Scientific and Technical Information of China (English)

    杨红; 路新卿; 李骥; 朱永健; 丁辉; 王健; 胡益群; 邓卫萍; 钱家鸣

    2013-01-01

    目的 观察ARHI基因转染PANC1细胞后对细胞凋亡和自噬相关基因mRNA表达谱的影响.方法 采用脂质体法将表达ARHI基因的质粒pIRES2-EGFP-ARHI、空质粒pIRES2-EGFP转染胰腺癌PANC1细胞.采用基因芯片RT2ProfilerTM PCR Array行实时定量PCR,分析转染细胞的基因表达谱,包括84个与凋亡和自噬相关基因.结果 ARHI基因转染组PANC1细胞有9个基因mRNA表达下调,38个基因mRNA表达上调,37个基因mRNA表达变化无意义.在与凋亡相关的基因中有8个促凋亡基因表达显著上调(>6倍),主要为TNFR/TRFSR家族基因(TNFSF8、TNFRSF10B、TNFRSF11B、TNFRSF9)、CIDE家族基因(DFFA)、CASP家族基因(CASP10、CASP8)和死亡结构域家族基因(DAPK1),其中以DAPK1上调尤为明显,达42.83倍;抗凋亡基因中3个基因(CD27、BCL2L10、BIRC4)表达显著上调(>6倍),3个基因(BCL2、BAD、BAG4)表达轻度上调(>2倍),1个基因(BCL2L1)表达轻度下调(<-2倍).在与自噬相关的基因中3个促自噬基因(TNFRSF10B、DAPK1、CASP10)表达显著上调(>6倍),4个基因(TNFRSF10A、FADD、TP53、TP53 BP2)表达轻度上调(>2倍);3个抑制自噬基因(BCL2、CASP8、FAS)表达轻度上调(>2倍),1个基因(MCL1)表达轻度下调(<-2倍).结论 ARHI基因显著上调细胞凋亡及自噬重要调控基因Caspase-8和DAPK1.%Objective To investigate the effect of ARHI transfection on the apoptosis and autophagy related gene expression profile of PANC1 cells.Methods Plasmids pIRES2-EGFP-ARHI which expressing ARHI and empty plasmid pIRES2-EGFP were transfected into PANC1 cells.Expression profile,including 84 apoptosis and autophagy related genes was detected by using quantitative real-time PCR based RT2Profiler TM PCR Array.Results In PANC1 cells transfected with pIRES2-EGFP-ARHI,the expression of mRNA of 9 genes were down-regulated,and 38 were up-regulated,while 37 were not significantly changed.Among the apoptosis related genes,8 pro-apoptotic genes were

  9. 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.

  10. Progress and application of salinomycin-induced cell autophagy in anti-cancer treatment%盐霉素诱导的细胞自噬机制在抗癌治疗中的应用进展

    Institute of Scientific and Technical Information of China (English)

    陈思远; 胡济安

    2016-01-01

    自噬在肿瘤的发生、发展、治疗过程中扮演着重要的角色。盐霉素作为一种聚醚类离子载体型抗生素,一直被用于防治鸡球虫病和促进反刍动物生长;它也能有效杀伤多种肿瘤干细胞及多药耐药肿瘤细胞系,且在此过程中伴随自噬的发生。盐霉素诱导的自噬是复杂的,可由细胞内活性氧升高而激活自噬;可通过损伤线粒体引发线粒体自噬;可引起内质网应激,通过 AKT1-mTOR信号通路激活自噬。此外,盐霉素可抑制自噬潮的晚期阶段,有助于肿瘤微环境的破坏和自身抗肿瘤作用的提高。本文综述了盐霉素在禽畜疾病中的应用及机制、盐霉素抗肿瘤的作用机制及盐霉素诱导自噬的机制。%Summary Autophagy is a cell self-eating process that allows the orderly degradation and recycling of misfolded proteins and damaged organelles through lysosome.It plays a vital role in cells”adaptation to metabolic stress and homeostasis as well as the formation,development and treatment of tumors.Autophagy is a double-edged sword, both suppressing and facilitating tumorigenesis.For example,multiple benign tumors formed in the liver when autophagy gene Atg5 was mosaically deleted in mice.But at the same time,cancer cells can upregulate autophagy to survive under microenvironmental stress, promote tumor dormancy and increase their growth and aggressiveness. Salinomycin is a polyether ionophore antibiotic isolated from Streptomycesalbus.Compared with lasalocid and monensin,salinomycin is less toxic,lower priced and less susceptible to drug resistance,which enables worldwide use to prevent chicken coccidiosis and promote the growth of ruminants.As an antimicrobial drug,salinomycin acts as an ionophore for K+ and Na+ ions and is able to alter cytoplasmic and mitochondrial membrane potentials, thereby inhibiting oxidative phosphorylation.Besides,salinomycin can activate a MutS homolog 1-dependent cell cycle

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

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-03-01

    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.

  12. Autophagy-deficiency in hepatic progenitor cells leads to the defects of stemness and enhances susceptibility to neoplastic transformation.

    Science.gov (United States)

    Xue, Feng; Hu, Lei; Ge, Ruiliang; Yang, Lixue; Liu, Kai; Li, Yunyun; Sun, Yanfu; Wang, Kui

    2016-02-01

    Autophagy is a highly conserved and lysosome-dependent degradation process which assists in cell survival and tissue homeostasis. Although previous reports have shown that deletion of the essential autophagy gene disturbs stem cell maintenance in some cell types such as hematopoietic and neural cells, it remains unclear how autophagy-deficiency influences hepatic progenitor cells (HPCs). Here we report that Atg5-deficiency in HPCs delays HPC-mediated rat liver regeneration in vivo. In vitro researches further demonstrate that loss of autophagy decreases the abilities of colony and spheroid formations, and disrupts the induction of hepatic differentiation in HPCs. Meanwhile, autophagy-deficiency increases the accumulations of damaged mitochondria and mitochondrial reactive oxygen species (mtROS) and suppresses homologous recombination (HR) pathway of DNA damage repair in HPCs. Moreover, in both diethylnitrosamine (DEN) and CCl4 models, autophagy-deficiency accelerates neoplastic transformation of HPCs. In conclusion, these findings demonstrate that autophagy contributes to stemness maintenance and reduces susceptibility to neoplastic transformation in HPCs.

  13. The role of autophagy as a mechanism of toxicity induced by multi-walled carbon nanotubes in human lung cells.

    Science.gov (United States)

    Tsukahara, Tamotsu; Matsuda, Yoshikaszu; Haniu, Hisao

    2014-12-23

    Carbon nanotubes (CNTs) are promising nanomaterials having unique physical and chemical properties, with applications in a variety of fields. In this review, we briefly summarize the intrinsic properties of highly purified multi-walled CNTs (MWCNTs, HTT2800) and their potential hazardous effects on intracellular and extracellular pathways, which alter cellular signaling and impact major cell functions such as differentiation, reactive oxygen species (ROS) production, apoptosis, and autophagy. A recent study suggested that the induction of autophagy by CNTs causes nanotoxicity. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with exposure to CNTs. Although autophagy is considered as a cytoprotective process, it is often observed in association with cell death, and the relationship between autophagy and cell death remains unclear. Our recent study suggests that the levels of autophagy-related genes (LC3B) and autophagosome formation are clearly up-regulated, along with an increase in numbers of autophagosome vacuoles. This review highlights the importance of autophagy as an emerging mechanism of CNT toxicity.

  14. Coffee induces autophagy in vivo

    Science.gov (United States)

    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 of LC3B and the reduction of the abundance of the autophagic substrate sequestosome 1 (p62/SQSTM1). These changes were accompanied by the inhibition of the enzymatic activity of mammalian target of rapamycin complex 1 (mTORC1), leading to the reduced phosphorylation of p70S6K, as well as by the global deacetylation of cellular proteins detectable by immunoblot. Immunohistochemical analyses of transgenic mice expressing a GFP–LC3B fusion protein confirmed the coffee-induced relocation of LC3B to autophagosomes, as well as general protein deacetylation. Altogether, these results indicate that coffee triggers 2 phenomena that are also induced by nutrient depletion, namely a reduction of protein acetylation coupled to an increase in autophagy. We speculate that polyphenols contained in coffee promote health by stimulating autophagy. PMID:24769862

  15. Coffee induces autophagy in vivo.

    Science.gov (United States)

    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 of LC3B and the reduction of the abundance of the autophagic substrate sequestosome 1 (p62/SQSTM1). These changes were accompanied by the inhibition of the enzymatic activity of mammalian target of rapamycin complex 1 (mTORC1), leading to the reduced phosphorylation of p70(S6K), as well as by the global deacetylation of cellular proteins detectable by immunoblot. Immunohistochemical analyses of transgenic mice expressing a GFP-LC3B fusion protein confirmed the coffee-induced relocation of LC3B to autophagosomes, as well as general protein deacetylation. Altogether, these results indicate that coffee triggers 2 phenomena that are also induced by nutrient depletion, namely a reduction of protein acetylation coupled to an increase in autophagy. We speculate that polyphenols contained in coffee promote health by stimulating autophagy.

  16. 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.

  17. 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

  18. Autophagy Pathway Is Required for IL-6 Induced Neuroendocrine Differentiation and Chemoresistance of Prostate Cancer LNCaP Cells

    Science.gov (United States)

    Chang, Yi-Ting; Chu, Cheng-Ying; Lee, Chin-Ling; Hsu, Hung-Wei; Zhou, Tyng-An; Wu, Zhaoju; Kim, Randie H.; Desai, Sonal J.; Liu, Shangqin; Kung, Hsing-Jien

    2014-01-01

    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 reveal the

  19. 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.

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

    Science.gov (United States)

    Bargiela, Ariadna; Cerro-Herreros, Estefanía; Fernandez-Costa, Juan M; Vilchez, Juan J; Llamusi, Beatriz; Artero, Ruben

    2015-07-01

    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.

  1. 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

  2. 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

  3. Cisplatin-induced apoptosis inhibits autophagy, which acts as a pro-survival mechanism in human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Barbara Del Bello

    Full Text Available The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology. In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains. The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response. Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin, protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing. In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains. On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms. In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.

  4. Keeping autophagy in cheCK1

    Science.gov (United States)

    Cheong, Jit Kong; Virshup, David M.

    2016-01-01

    Abstract Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1α), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RAS-induced autophagy and the development of mutant RAS-driven cancers. PMID:27314070

  5. 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

  6. Regulation of autophagy by cytoplasmic p53.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  7. 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.

  8. Midgut morphological changes and autophagy during metamorphosis in sand flies.

    Science.gov (United States)

    Malta, Juliana; Heerman, Matthew; Weng, Ju Lin; Fernandes, Kenner M; Martins, Gustavo Ferreira; Ramalho-Ortigão, Marcelo

    2017-03-11

    During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

  9. Autophagy is upregulated during colorectal carcinogenesis, and in DNA microsatellite stable carcinomas.

    Science.gov (United States)

    Sena, Paola; Mariani, Francesco; Mancini, Stefano; Benincasa, Marta; Magnani, Giulia; Pedroni, Monica; Palumbo, Carla; Roncucci, Luca

    2015-12-01

    Cancer cells are exposed to a wide range of stress sources, such as nutrient deprivation and hypoxia, as well as cytotoxic chemotherapy and radiotherapy. Certain forms of stress can also promote survival activating the metabolic autophagy pathway in cancer cells. Autophagy is dramatically increased in cancer cells. In these conditions, it is becoming evident that autophagy protects cells, by providing an alternative energy source and by eliminating dysfunctional organelles or proteins. Its role in tumorigenesis is more controversial and both the presence and the absence of autophagy have been implicated. Autophagy is known to be associated with the poor outcome of patients with various types of cancers, and its effectiveness as a prognostic marker in colorectal cancer was demonstrated by several studies. The inhibition of autophagy may be a potential therapeutic target in colorectal cancer. In vitro experiments have shown that the inhibition of autophagy increases 5-FU-induced apoptosis. There are two trials currently investigating the addition of chloroquine to 5-FU-based chemotherapy and bevacizumab. In the present study, we evaluated the expression of LC3B-II in samples of human colorectal microadenomas (i.e., dysplastic aberrant crypt foci) and carcinomas compared to normal mucosa. Furthermore, the expression pattern of LC3B-II was assessed in carcinomas classified as DNA microsatellite stable (MSS) and unstable (MSI). Thus, immunofluorescence techniques coupled with confocal microscopy and immunoblot experiments were performed. The results clearly showed a significant increase in expression of the autophagic key factor in microadenomas and carcinomas with respect to normal mucosa. In MSS carcinomas, the level of LC3B-II expression was higher than that in the MSI carcinomas.

  10. Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

    Science.gov (United States)

    Buckingham, Erin M.; Jarosinski, Keith W.; Jackson, Wallen; Carpenter, John E.

    2016-01-01

    intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells. PMID:27440906

  11. 细胞自噬的研究方法%Methods for Autophagy Detection

    Institute of Scientific and Technical Information of China (English)

    马泰; 孙国平; 李家斌

    2012-01-01

    Autophagy extensively participate in physiological and pathological processes, and has been focused by contemporary biomedicine scientists in recent years. Transmission electron microscopy, immunofluorescence and immunoblotting techniques were common used in detection of autophagy. Deeper research needs more accurate detection of autophagy. Dysfunction of autophagy involves formation and degeneration of autophosome, accordingly, accurate and comprehensive evaluation of autophagy includes autophosome detection, as well as the fluency of autophagic degeneration, I.e. Autophagic flux assay. Additionally, artificial up- or down-regulation of autophagy by drugs or gene interferences in in vitro or in vivo models has also been considered as important part of autophagy analysis. Any method currently used alone may not been as evidence of autophagy. More careful attention should be paid on results of any assays of autophagy, especially DO NOT interpret "increase or decrease of autophosome" (also "up- or down-expression of autophagy-related proteins") as "enhancement or attenuation of autophagic function".%细胞自噬的研究是目前生物医学领域热点之一,广泛参与各种生理和病理过程.目前普遍采用的自噬检测方法包括电镜、免疫荧光、蛋白质印迹等方法检测自噬体及其标志蛋白.研究的深入对自噬的检测方法也提出了更高的要求,自噬功能障碍包括自噬体形成和降解障碍,因此,准确全面地评估自噬不仅包括自噬体的检测,还包括动态观察整个自噬性降解的过程是否顺畅(即自噬潮分析).另外,通过药物或基因干预技术来人为地调控自噬以观察其在体内体外模型中的作用也是自噬分析的重要内容.需要注意的是,任何一种方法单独应用均不能作为自噬的依据,对任何方法得到的结果进行解释时必须慎重,特别是不能将自噬体的增多减少或自噬相关蛋白表达的高低等同于自噬的增强或减弱.

  12. Mechanisms of mitochondria and autophagy crosstalk.

    Science.gov (United States)

    Rambold, Angelika S; Lippincott-Schwartz, Jennifer

    2011-12-01

    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 neuronal loss in Parkinson's disease. In addition to autophagy's significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria's ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.

  13. 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.

  14. Emerging connections between RNA and autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lubas, Michal; Lund, Anders H

    2016-01-01

    Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority...... of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome....../vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNA-related functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy...

  15. 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.