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Sample records for beclin 1-dependent autophagy

  1. β-asarone improves learning and memory and reduces Acetyl Cholinesterase and Beta-amyloid 42 levels in APP/PS1 transgenic mice by regulating Beclin-1-dependent autophagy.

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    Deng, Minzhen; Huang, Liping; Ning, Baile; Wang, Nanbu; Zhang, Qinxin; Zhu, Caixia; Fang, Yongqi

    2016-12-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, and studies have suggested that β-asarone has pharmacological effects on beta-amyloid (Aβ) injected in the rat hippocampus. However, the effect of β-asarone on autophagy in the APP/PS1 transgenic mouse is unreported. APP/PS1 transgenic mice were randomly divided into six groups (n=10/group): an untreated group, an Aricept-treated group, a 3-MA-treated group, a rapamycin-treated group, an LY294002-treated group, a β-asarone-treated group. The control group consisted of wild-type C57BL/6 mice. All treatments were administered to the mice for 30 days. Spatial learning and memory were assessed by water maze, passive avoidance, and step-down tests. AChE and Aβ 42 levels in the hippocampus were determined by ELISA. p-Akt, p-mTOR, and LC3B expression were detected by flow cytometry. The expression of p-Akt, p-mTOR, Beclin-1, and p62 proteins was assessed by western blot. Changes in autophagy were viewed using a transmission electron microscope. APP and Beclin-1 mRNA levels were measured by Real-Time PCR. The learning and memory of APP/PS1 transgenic mice were improved significantly after β-asarone treatment compared with the untreated group. In addition, β-asarone treatment reduced AChE and Aβ 42 levels, increased p-mTOR and p62 expression, decreased p-Akt, Beclin-1, and LC3B expression, decreased the number of autophagosomes and reduced APP mRNA and Beclin-1 mRNA levels compared with the untreated group. That is, β-asarone treatment can improve the learning and memory abilities of APP/PS1 transgenic mouse by inhibiting Beclin-1-dependent autophagy via the PI3K/Akt/mTOR pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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    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.

  3. Expression and Clinical Significance of the Autophagy Proteins BECLIN 1 and LC3 in Ovarian Cancer

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    Guido Valente

    2014-01-01

    Full Text Available Autophagy is dysregulated in cancer and might be involved in ovarian carcinogenesis. BECLIN-1, a protein that interacts with either BCL-2 or PI3k class III, plays a critical role in the regulation of both autophagy and cell death. Induction of autophagy is associated with the presence of vacuoles characteristically labelled with the protein LC3. We have studied the biological and clinical significance of BECLIN 1 and LC3 in ovary tumours of different histological types. The positive expression of BECLIN 1 was well correlated with the presence of LC3-positive autophagic vacuoles and was inversely correlated with the expression of BCL-2. The latter inhibits the autophagy function of BECLIN 1. We found that type I tumours, which are less aggressive than type II, were more frequently expressing high level of BECLIN 1. Of note, tumours of histologic grade III expressed low level of BECLIN 1. Consistently, high level of expression of BECLIN 1 and LC3 in tumours is well correlated with the overall survival of the patients. The present data are compatible with the hypotheses that a low level of autophagy favours cancer progression and that ovary cancer with upregulated autophagy has a less aggressive behaviour and is more responsive to chemotherapy.

  4. Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction.

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    Zhou, Ao; Li, Shuaifeng; Khan, Faheem Ahmed; Zhang, Shujun

    2016-01-01

    Autophagy and apoptosis play significant roles in PRRSV infection and replication. However, the interaction between these 2 processes in PRRSV replication is still far from been completely understood. In our studies, the exposure of MARC-145 cells to PRRSV confirmed the activation of autophagy and subsequent induction of apoptosis. The inhibition of autophagy by 3-methyladenine (3-MA) caused a significant increase in PRRSV-induced apoptosis, showing a potential connection between both mechanisms. Moreover, we observed an increase in Bad expression (a pro-apoptotic protein) and Beclin1 (an autophagy regulator) in virus-infected cells up to 36h. Co-immunoprecipitation assays showed the formation of Bad and Beclin1 complex in PRRSV infected cells. Accordingly, Bad co-localized with Beclin1 in MARC-145 infected cells. Knockdown of Beclin1 significantly decreased PRRSV replication and PRRSV-induced autophagy, while Bad silencing resulted in increased autophagy and enhanced viral replication. Furthermore, PRRSV infection phosphorylated Bad (Ser112) to promote cellular survival. These results demonstrate that autophagy can favor PRRSV replication by postponing apoptosis through the formation of a Bad-Beclin1 complex.

  5. Effect of baicalin on the autophagy and Beclin-1 expression in rats with cerebral ischemia

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    Xiang-Long Hong

    2016-07-01

    Full Text Available Objective: To explore the effect of baicalin on the autophagy and Beclin-1 expression in rats with cerebral ischemia, and the role of autophagy in the cerebral ischemia injury. Methods: The healthy male SD rats were randomized into the sham operation group, the ischemia model group, baicalin treatment group (100 mg/kg, and 3MA group (15 mg/kg, with 10 rats in each group. Transient focal cerebral ischemia injury model in rats was induced by occlusion of middle cerebral artery (MCA for 180 min. The rats were given the corresponding drugs through the tail veins 30 min before molding. Half of the specimens were used for TTC staining to analyze the cerebral infarction volume. The others were used to determine the expression of Beclin-1 in the brain tissues by Western-blot. Results: When compared with the ischemia model group, the cerebral infarction volume in 3MA group was significantly increased, while that in baicalin treatment group was significantly reduced, and the comparison among the groups was statistically significant. When compared with the ischemia model group, Beclin-1 expression level in baicalin treatment group was significantly elevated, while Beclin-1 expression level in 3MA group was significantly higher than that in the sham-operation group but lower than that in the ischemia model group. Conclusions: The autophagy level of brain tissues in normal rats is low. The cerebral ischemia can activate autophagy. The activated autophagy is probably involved in the neuroprotection of cerebral ischemia injury. Application of 3MA to inhibit the occurrence of autophagy can aggravate the cerebral injury. Baicalin can significantly improve the cerebral ischemia injury and promote the occurrence of autophagy, whose mechanism is probably associated with the up-regulation of Beclin-1 expression to promote the activation of type III PI3K signal transduction pathway.

  6. The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced autophagy versus apoptosis in prostate cancer cells.

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    Lian, Jiqin; Karnak, David; Xu, Liang

    2010-11-01

    Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

  7. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

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    Tian, Ping-Ge [Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Zhi-Xin [Centre Laboratory, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China); Li, Jian-Hua [Department of Geriatric Cardiology, Chinese PLA General Hosptial, Beijing 100853 (China); Zhou, Zhe, E-mail: zhouzhe76@126.com [Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Zhang, Qing-Hua, E-mail: 1056055170@qq.com [Department of Cardiology, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China)

    2015-08-07

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

  8. Carnosol induces ROS-mediated beclin1-independent autophagy and apoptosis in triple negative breast cancer.

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    Yusra Al Dhaheri

    Full Text Available In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2. Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.

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

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    Chang, Seung-Hee; Minai-Tehrani, Arash; Shin, Ji-Young

    2012-01-01

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

  10. Structural transitions in conserved, ordered Beclin 1 domains essential to regulating autophagy

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    Glover, Karen; Li, Yue; Mukhopadhyay, Shreya; Leuthner, Zoe; Chakravarthy, Srinivas; Colbert, Christopher L.; Sinha, Sangita C. (NDSU); (IIT)

    2017-08-10

    Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositol-3-kinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiled-coil domain (CCD). However, the last 16 CCD residues, composing an “overlap helix” (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the C-terminal β-α repeated, autophagy-specific domain (BARAD). Here, using CD spectroscopy, isothermal titration calorimetry, and small-angle X-ray scattering, we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the two-domain CCD–BARAD. Last, we used cellular assays to demonstrate that mutation of these OH-interface residues abrogates starvation-induced up-regulation of autophagy but does not affect basal autophagy. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains (i.e. the CCD or the BARAD). Our findings have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes.

  11. Structural insights into the interaction of the conserved mammalian proteins GAPR-1 and Beclin 1, a key autophagy protein

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    Li, Yue; Zhao, Yuting; Su, Minfei; Glover, Karen; Chakravarthy, Srinivas; Colbert, Christopher L.; Levine, Beth; Sinha, Sangita C.

    2017-08-29

    Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267–284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1–GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide. A model of a peptide comprising Beclin 1 residues 267–284 docked onto GAPR-1, built using theCABS-dockserver, indicates that this peptide binds to this GAPR-1 groove. Mutation of the five conserved residues lining this groove, H54A/E86A/G102K/H103A/N138G, abrogates Beclin 1 binding. The 1.27 Å resolution X-ray crystal structure of this pentad mutant GAPR-1 was determined. Comparison with the wild-type (WT) GAPR-1 structure shows that the equatorial groove of the pentad mutant is shallower and more positively charged, and therefore may not efficiently bind Beclin 1 residues 267–284, which include many hydrophobic residues. Both WT and pentad mutant GAPR-1 crystallize as dimers, and in each case the equatorial groove of one subunit is partially occluded by the other subunit, indicating that dimeric GAPR-1 is unlikely to bind Beclin 1. SAXS analysis of WT and pentad mutant GAPR-1 indicates that in solution the WT forms monomers, while the pentad mutant is primarily dimeric. Thus, changes in the structure of the equatorial groove combined with the improved dimerization of pentad mutant GAPR-1 are likely to abrogate binding to Beclin 1.

  12. miR-409-3p sensitizes colon cancer cells to oxaliplatin by inhibiting Beclin-1-mediated autophagy.

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    Tan, Shifan; Shi, Huijuan; Ba, Mingchen; Lin, Shengqv; Tang, Hongsheng; Zeng, Xiaoqi; Zhang, Xiangliang

    2016-04-01

    The chemoresistance of colon cancer cells limits the efficacy of chemotherapy. miR-409-3p has been shown to be downregulated in various types of cancer. In the present study, we examined the role of miR-409-3p in colon cancer as well as the effects of miR‑409-3p on the sensitivity of colon cancer cells to oxaliplatin. The expression of miR-409 was significantly downregulated in the human colon cancer cell lines compared with the normal colon epithelial cells. Importantly, the miR-409-3p expression levels were lower in human colon cancer patient samples than in normal colon tissues. Moreover, we observed a negative correlation between the miR‑409-3p levels and resistance to oxaliplatin: the oxaliplatin-resistant colon cancer cells exhibited significantly downregulated miR‑409-3p levels, but higher autophagic activity than the oxaliplatin-sensitive cells. Using bioinformatics analysis, we predicted that miR‑409-3p miRNA binds to the key autophagy gene encoding Beclin-1. Our findings indicated that the overexpression of miR‑409-3p inhibited Beclin-1 expression and autophagic activity by binding to the 3'-untranslated region of Beclin-1 mRNA. In addition, the overexpression of miR‑409-3p enhanced the chemosensitivity of the oxaliplatin-sensitive and oxaliplatin-resistant colon cancer cells. The restoration of Beclin-1 abrogated these effects of miR‑409-3p. In a xenograft model using nude mice, we examined the effects of miR‑409-3p on tumor growth during chemotherapy. miR‑409-3p overexpression sensitized the tumor to chemotherapy, while inhibiting chemotherapy-induced autophagy in a manner dependent on Beclin-1. The findings of our study suggest that miR-409-3p is capable of enhancing the chemosensitivity of colon cancer cells by inhibiting Beclin-1-mediated autophagy.

  13. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L).

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    Maiuri, Maria Chiara; Criollo, Alfredo; Tasdemir, Ezgi; Vicencio, José Miguel; Tajeddine, Nicolas; Hickman, John A; Geneste, Olivier; Kroemer, Guido

    2007-01-01

    Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.

  14. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells.

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    Scarlatti, F; Maffei, R; Beau, I; Codogno, P; Ghidoni, R

    2008-08-01

    Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

  15. Drp1-Dependent Mitochondrial Autophagy Plays a Protective Role Against Pressure Overload-Induced Mitochondrial Dysfunction and Heart Failure.

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    Shirakabe, Akihiro; Zhai, Peiyong; Ikeda, Yoshiyuki; Saito, Toshiro; Maejima, Yasuhiro; Hsu, Chiao-Po; Nomura, Masatoshi; Egashira, Kensuke; Levine, Beth; Sadoshima, Junichi

    2016-03-29

    Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood. Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at ≈3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy. Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload. © 2016 American Heart Association, Inc.

  16. Expressions and clinical significance of autophagy-related markers Beclin1, LC3, and EGFR in human cervical squamous cell carcinoma

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    Hu YF

    2015-08-01

    Full Text Available Yun-Feng Hu,1 Xia Lei,2 Hong-Yi Zhang,3 Jun-wei Ma,1 Wei-wei Yang,1 Min-lin Chen,1 Jie Cui,1,4 Hong Zhao1 1Department of Oncology, 2Department of Gynecology, 3Department of Urology, Yan’an University Affiliated Hospital, Yan’an, Shaanxi Province, People’s Republic of China; 4Department of Oncology, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, People’s Republic of China Purpose: We aimed to investigate the expression of EGFR and the autophagy-related markers Beclin1 and LC3 in cervical cancer.Methods: Beclin1, LC3, and EGFR expression were analyzed in 80 samples of cervical squamous cell carcinoma (SCC, 40 samples of high-grade cervical intraepithelial neoplasia (CIN, and 40 samples of normal cervical tissues by immunohistochemistry. The protein expression rates were analyzed with χ2 and Fisher’s exact tests. Differences in overall survival (OS were determined using the Kaplan–Meier method and log-rank tests.Results: Cervical cancer, high-grade CIN, and normal cervical epithelial cells expressed Beclin1 in 26.2%, 77.5%, and 82.5% of patients, respectively, and expressed LC3 in 28.8%, 70.0%, and 75.0% of patients, respectively. There was a significant difference between cervical SCC and high-grade CIN or normal cervical epithelial cells (P=0.000. Cervical cancer cells, high-grade CIN cells, and normal cervical epithelial cells expressed EGFR in 68.8%, 62.5%, and 12.5% of patients, respectively. There was a significant difference between cervical SCC or high-grade CIN and normal cervical epithelial cells (P=0.000. No significant association between Beclin1 or LC3 or EGFR expression and various clinicopathological parameters was observed in cervical SCC. There was no significant correlation between Beclin1, LC3, EGFR expression, and 5-year OS rates of cervical SCC patients. Beclin1- or LC3-negativity with EGFR-positivity in cervical SCC was associated with a higher Federation International of

  17. Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress.

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    Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R; Scott, Melanie J

    2013-05-31

    Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.

  18. Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress*

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    Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R.; Scott, Melanie J.

    2013-01-01

    Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed. PMID:23589298

  19. Direct molecular interactions between Beclin 1 and the canonical NFκB activation pathway.

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    Niso-Santano, Mireia; Criollo, Alfredo; Malik, Shoaib Ahmad; Michaud, Michael; Morselli, Eugenia; Mariño, Guillermo; Lachkar, Sylvie; Galluzzi, Lorenzo; Maiuri, Maria Chaira; Kroemer, Guido

    2012-02-01

    General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.

  20. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

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    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

  1. The prognostic value of autophagy-related markers beclin-1 and microtubule-associated protein light chain 3B in cancers: a systematic review and meta-analysis.

    Science.gov (United States)

    He, Yuyu; Zhao, Xianda; Subahan, Narishka Roz; Fan, Lifang; Gao, Jun; Chen, Honglei

    2014-08-01

    Use of the autophagy-related markers beclin-1 (BECN1) and microtubule-associated protein light chain 3B (LC3B) as prognostic markers has been extensively investigated in various kinds of cancers. However, their prognostic roles are still controversial and not firmly validated. We systematically reviewed the evidence from various studies concerning the relationship between BECN1 and LC3B expression in cancers and overall survival (OS)/disease-free survival (DFS) to elucidate this issue. PubMed, Embase, Cochrane Library, and China National Knowledge Infrastructure (CNKI) were searched in July 2013 (then updated in April 2014) to identify eligible cohort studies that reported associations between BECN1 or LC3B expression and OS/DFS in cancer patients. Combined hazard ratios (HRs) with 95 % confidence intervals (95 % CIs) were pooled using fixed-effects or random-effects models according to heterogeneity in different groups. A total of 23 studies in distinct cancers were eligible for systematic review and meta-analysis. Our pooled results identified that a high expression of BECN1 is associated with favorable OS in gastric cancer (HR = 0.49, 95 % CI = 0.34-0.72) and lymphoma (HR = 0.25, 95 % CI = 0.11-0.57), whereas a high expression of LC3B predicts adverse OS in breast cancer (HR = 1.98, 95 % CI = 1.25-3.13). This systematic review and meta-analysis indicated that the autophagy-related marker BECN1 might be a predictive factor of favorable prognosis in gastric cancer, breast cancer, and lymphoma and LC3B might predict unfavorable prognosis of breast cancer. Nevertheless, due to the limited number and retrospective design of the original studies, more powerful prospective cohorts are required to verify these conclusions.

  2. Single-Prolonged-Stress-Induced Changes in Autophagy-Related Proteins Beclin-1, LC3, and p62 in the Medial Prefrontal Cortex of Rats with Post-traumatic Stress Disorder.

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    Zheng, Shilei; Han, Fang; Shi, Yuxiu; Wen, Lili; Han, Dan

    2017-05-01

    Autophagy, or type II programmed cell death, plays a crucial role in many nervous system diseases. However, few studies have examined the role of autophagy in post-traumatic stress disorder (PTSD), and the mechanisms underlying PTSD are poorly understood. The objective of this research was to explore the expression of three important autophagy-related proteins, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and p62/SQSTM1 (p62), in the medial prefrontal cortex (mPFC) of an animal model of PTSD to identify changes in autophagic activity during PTSD pathogenesis. PTSD was induced in rats by exposure to a single-prolonged stress (SPS). The Morris water maze was used to assess cognitive changes in rats from the SPS and control groups. Transmission electron microscopy (TEM) was employed to observe mPFC morphological changes. Immunohistochemistry, immunofluorescence, and Western blotting techniques were used to detect expression of Beclin-1, LC3, and p62 in the mPFC. The Morris water maze test results showed that the escape latency time was increased and that the percent time in the target quadrant was decreased in the SPS group compared with that in the control group. Numerous visible autolysosomes in mPFC neurons were observed using TEM after SPS stimulation. Compared with that in the control group, the expression of Beclin-1 and the LC3-II/I ratio significantly decreased at 1 day, then increased and peaked at 7 days, and slightly decreased at 14 days after SPS stimulation, whereas the converse was found for p62 expression. In conclusion, dysregulation of autophagic activity in the mPFC may play a crucial role in PTSD pathogenesis.

  3. Drp-1 dependent mitochondrial fragmentation and protective autophagy in dopaminergic SH-SY5Y cells overexpressing alpha-synuclein.

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    Martinez, Jimena Hebe; Alaimo, Agustina; Gorojod, Roxana Mayra; Porte Alcon, Soledad; Fuentes, Federico; Coluccio Leskow, Federico; Kotler, Mónica Lidia

    2018-04-01

    Parkinson's disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. Additional common features of this disease are the mitochondrial dysfunction and the activation/inhibition of autophagy both events associated to the intracellular accumulation of α-synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of α-synuclein on mitochondrial dynamics and autophagy/mitophagy in SH-SY5Y cells, an in vitro model of Parkinson disease. We demonstrated that overexpression of wild type α-synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, α-synuclein induces the mitochondrial fragmentation on a Drp-1-dependent fashion. Overexpression of the fusion protein Opa-1 prevented both mitochondrial fragmentation and cytotoxicity. On the other hand, cells expressing α-synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from α-synuclein-induced cell death. Our results clarify the role of Opa-1 and Drp-1 in mitochondrial dynamics and cell survival, a controversial α-synuclein research issue. The findings presented point to the relevance of mitochondrial homeostasis and autophagy in the pathogenesis of PD. Better understanding of the molecular interaction between these processes could give rise to novel therapeutic methods for PD prevention and amelioration. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas

    International Nuclear Information System (INIS)

    Hasui, Kazuhisa; Wang, Jia; Jia, Xinshan; Tanaka, Masashi; Nagai, Taku; Matsuyama, Takami; Eizuru, Yoshito

    2011-01-01

    This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV + lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV + NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL

  5. Resveratrol alleviates cerebral ischemia/reperfusion injury in rats by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy induction.

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    He, Qi; Li, Zhenyu; Wang, Yueting; Hou, Yanghao; Li, Lingyu; Zhao, Jing

    2017-09-01

    Resveratrol has been reported to protect against cerebral ischemia/reperfusion (I/R) injury in rats, but the underlying mechanism is unclear. In the current study, we examined whether resveratrol ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome-derived inflammation and whether autophagy is involved in this process. In addition, we explored the role of Sirt1 in resveratrol-mediated protective effects. To answer these questions, healthy male Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 1h followed by 24h reperfusion. We found that cerebral I/R increased levels of activated NLRP3 inflammasome, caspase-1, IL-1β, and IL-18 and enhanced autophagy activity (ratio of LC3B-II/LC3B-I and p62/SQSTM1). Treatment with resveratrol, a specific Sirt1 agonist, attenuated I/R-induced NLRP3 inflammasome-derived inflammation but upregulated autophagy. Furthermore, resveratrol treatment clearly reduced cerebral infarct volume, decreased brain water content, and improved neurological scores. In addition, inhibition of autophagy using 3-MA intracerebroventricular injection blocked the inhibitory effect of resveratrol on NLRP3 inflammasome activation. Finally, Sirt1 knockdown with siRNA significantly blocked resveratrol-induced enhancement of autophagy activity and suppression of NLRP3 inflammasome activation. In conclusion, our results demonstrate that resveratrol protects against cerebral I/R injury by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy activity. Copyright © 2017. Published by Elsevier B.V.

  6. VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

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    Qian, Qinyi [Department of Ultrasonograph, Changshu No. 2 People’s Hospital, Changshu (China); Zhou, Hao; Chen, Yan [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China); Shen, Chenglong [Department of General Surgery, Changshu No. 2 People’s Hospital, Changshu (China); He, Songbing; Zhao, Hua; Wang, Liang [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China); Wan, Daiwei, E-mail: 372710369@qq.com [Department of Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou (China); Gu, Wen, E-mail: 505339704@qq.com [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China)

    2014-01-17

    Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

  7. Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner.

    Science.gov (United States)

    Man, Na; Tan, Yurong; Sun, Xiao-Jian; Liu, Fan; Cheng, Guoyan; Greenblatt, Sarah M; Martinez, Camilo; Karl, Daniel L; Ando, Koji; Sun, Ming; Hou, Dan; Chen, Bingyi; Xu, Mingjiang; Yang, Feng-Chun; Chen, Zhu; Chen, Saijuan; Nimer, Stephen D; Wang, Lan

    2017-05-18

    AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease. © 2017 by The American Society of Hematology.

  8. Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

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    Defeng Wu

    2013-01-01

    Full Text Available Autophagy has been shown to be protective against drug and alcohol-induced liver injury. CYP2E1 plays a role in the toxicity of ethanol, carcinogens and certain drugs. Inhibition of autophagy increased ethanol-toxicity and accumulation of fat in wild type and CYP2E1 knockin mice but not in CYP2E1 knockout mice as well as in HepG2 cells expressing CYP2E1 (E47 cells but not HepG2 cells lacking CYP2E1 (C34 cells. The goal of the current study was to evaluate whether modulation of autophagy can affect CYP2E1-dependent cytotoxicity in the E47 cells. The agents used to promote CYP2E1 –dependent toxicity were a polyunsaturated fatty acid, arachidonic acid (AA, buthionine sulfoximine (BSO, which depletes GSH, and CCl4, which is metabolized to the CCl3 radical. These three agents produced a decrease in E47 cell viability which was enhanced upon inhibition of autophagy by 3-methyladenine (3-MA or Atg 7 siRNA. Toxicity was lowered by rapamycin which increased autophagy and was much lower to the C34 cells which do not express CYP2E1. Toxicity was mainly necrotic and was associated with an increase in reactive oxygen production and oxidative stress; 3-MA increased while rapamycin blunted the oxidative stress. The enhanced toxicity and ROS formation produced when autophagy was inhibited was prevented by the antioxidant N-Acetyl cysteine. AA, BSO and CCl4 produced mitochondrial dysfunction, lowered cellular ATP levels and elevated mitochondrial production of ROS. This mitochondrial dysfunction was enhanced by inhibition of autophagy with 3-MA but decreased when autophagy was increased by rapamycin. The mitogen activated protein kinases p38 MAPK and JNK were activated by AA especially when autophagy was inhibited and chemical inhibitors of p38 MAPK and JNK lowered the elevated toxicity of AA produced by 3-MA. These results show that autophagy was protective against the toxicity produced by several agents known to be activated by CYP2E1. Since CYP2E1 plays an

  9. Beclin 1 and UVRAG confer protection from radiation-induced DNA damage and maintain centrosome stability in colorectal cancer cells.

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    Jae Myung Park

    Full Text Available Beclin 1 interacts with UV-irradiation-resistance-associated gene (UVRAG to form core complexes that induce autophagy. While cells with defective autophagy are prone to genomic instability that contributes to tumorigenesis, it is unknown whether Beclin1 or UVRAG can regulate the DNA damage/repair response to cancer treatment in established tumor cells. We found that siRNA knockdown of Beclin 1 or UVRAG can increase radiation-induced DNA double strand breaks (DSBs, shown by pATM and γH2Ax, and promote colorectal cancer cell death. Furthermore, knockdown of Beclin 1, UVRAG or ATG5 increased the percentage of irradiated cells with nuclear foci expressing 53BP1, a marker of nonhomologous end joining but not RAD51 (homologous recombination, compared to control siRNA. Beclin 1 siRNA was shown to attenuate UVRAG expression. Cells with a UVRAG deletion mutant defective in Beclin 1 binding showed increased radiation-induced DSBs and cell death compared to cells with ectopic wild-type UVRAG. Knockdown of Beclin 1 or UVRAG, but not ATG5, resulted in a significant increase in centrosome number (γ-tubulin staining in irradiated cells compared to control siRNA. Taken together, these data indicate that Beclin 1 and UVRAG confer protection against radiation-induced DNA DSBs and may maintain centrosome stability in established tumor cells.

  10. Pravastatin Protects Against Avascular Necrosis of Femoral Head via Autophagy.

    Science.gov (United States)

    Liao, Yun; Zhang, Ping; Yuan, Bo; Li, Ling; Bao, Shisan

    2018-01-01

    Autophagy serves as a stress response and may contribute to the pathogenesis of avascular necrosis of the femoral head induced by steroids. Statins promote angiogenesis and ameliorate endothelial functions through apoptosis inhibition and necrosis of endothelial progenitor cells, however the process used by statins to modulate autophagy in avascular necrosis of the femoral head remains unclear. This manuscript determines whether pravastatin protects against dexamethasone-induced avascular necrosis of the femoral head by activating endothelial progenitor cell autophagy. Pravastatin was observed to enhance the autophagy activity in endothelial progenitor cells, specifically by upregulating LC3-II/Beclin-1 (autophagy related proteins), and autophagosome formation in vivo and in vitro . An autophagy inhibitor, 3-MA, reduced pravastatin protection in endothelial progenitor cells exposed to dexamethasone by attenuating pravastatin-induced autophagy. Adenosine monophosphate-activated protein kinase (AMPK) is a key autophagy regulator by sensing cellular energy changes, and indirectly suppressing activation of the mammalian target of rapamycin (mTOR). We found that phosphorylation of AMPK was upregulated however phosphorylation of mTOR was downregulated in pravastatin-treated endothelial progenitor cells, which was attenuated by AMPK inhibitor compound C. Furthermore, liver kinase B1 (a phosphorylase of AMPK) knockdown eliminated pravastatin regulated autophagy protein LC3-II in endothelial progenitor cells in vitro . We therefore demonstrated pravastatin rescued endothelial progenitor cells from dexamethasone-induced autophagy dysfunction through the AMPK-mTOR signaling pathway in a liver kinase B1-dependent manner. Our results provide useful information for the development of novel therapeutics for management of glucocorticoids-induced avascular necrosis of the femoral head.

  11. Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors

    International Nuclear Information System (INIS)

    Li, Zidong; Chen, Bo; Wu, Yiqing; Jin, Feng; Xia, Yongjing; Liu, Xiangjun

    2010-01-01

    Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer

  12. Intensified Beclin-1 Mediated by Low Expression of Mir-30a-5p Promotes Chemoresistance in Human Small Cell Lung Cancer

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    Xiang Yang

    2017-10-01

    Full Text Available Background/Aims: Although small cell lung cancer (SCLC is sensitive to initial chemotherapy, patients experience tumor recurrence and metastasis, leading to treatment failure. Autophagy as a protective pattern for cell survival in the harsh environment plays an important role in chemoresistance. However, the role of Beclin-1, a key regulator of autophagy in the drug-resistance of SCLC cells is still poorly understood. In the current study, we focused on the effect and regulation of Beclin-1 in chemoresistance of SCLC cells. Methods: We analyzed the levels of Beclin-1 in etoposide/cisplatin (EP -resistant and -sensitive cell lines, as well as the relationship between Beclin-1 and patients’ chemosensitivity. The function of Beclin-1 in chemoresistant SCLC cells in vitro was measured by MTT, WB, colony formation and flow cytometric analysis. Further rescue experiment was performed after co-transfected with siBeclin-1 and miR-30a mimics or inhibitor. Results: Beclin-1 was upregulated in drug-resistant cells and patients with lower sensitivity to etoposide/cisplatin therapy. Downregulated Beclin-1 attenuated drug sensitivity and colony formation ability of chemoresistant cells. Moreover, inhibition of Beclin-1 resulted in a dramatic decline of autophagy and increase of apoptosis in drug-resistant cells, accompanied by a remarkable reduction in S phase and a raise in G2/M phase of cell cycle. The transfection with miR-30a-5p mimics exhibited an opposite effect. In addition, inhibition of Beclin-1 could partly reverse the effect induced by miR-30a-5p suppression in drug-sensitive cells. Conclusion: Beclin-1 regulated by miR-30a-5p plays a notable role in the drug-resistance of SCLC. Inhibition of Beclin-1 by induction of miR-30a-5p may improve the therapeutic outcome via resensitizing the drug-resistant cells to chemotherapy in SCLC.

  13. Beclin 1 Expression is Closely Linked to Colorectal Carcinogenesis and Distant Metastasis of Colorectal Carcinoma

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    Mei-Ying Zhang

    2014-08-01

    Full Text Available Beclin 1 participates in development, autophagy, differentiation, anti- apoptosis, neurodegeneration, tumorigenesis and cancer progression. The roles of Beclin 1 in colorectal carcinogenesis and its subsequent progression are still unclear. Here, the mRNA and protein expression of Beclin 1 were determined in colorectal carcinoma and matched mucosa by Reverse transcriptase-polymerase chain reaction and Western blot. Immunohistochemistry and in situ hybridization (ISH were performed on tissue microarryer with colorectal carcinoma, adenoma and mucosa. The expression of Beclin 1 mRNA and protein was found to be higher in colorectal carcinoma than matched mucosa by real-time PCR and Western blot (p < 0.05. According to the ISH data, Beclin 1 expression was lower in colorectal non-neoplastic mucosa (NNM than adenoma and carcinoma (p < 0.05. Immunohistochemically, primary carcinoma showed stronger Beclin 1 expression than NNM and metastatic carcinoma in the liver (p < 0.05. Beclin 1 protein expression was negatively related to liver and distant metastasis (p < 0.05, but not correlated with age, sex, depth of invasion, lymphatic or venous invasion, lymph node metastasis, tumor-node-metastasis (TNM staging, differentiation or serum carcinoembryonic antigen (CEA concentration (p > 0.05. Survival analysis indicated that Beclin 1 expression was not linked to favorable prognosis of the patients with colorectal carcinoma (p > 0.05. Cox’s model indicated that depth of invasion and distant metastasis were independent prognostic factors for colorectal carcinomas (p < 0.05. It was suggested that Beclin 1 expression is closely linked to colorectal carcinogenesis and distant metastasis of colorectal carcinoma.

  14. Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver.

    Science.gov (United States)

    Petrović, Anja; Bogojević, Desanka; Korać, Aleksandra; Golić, Igor; Jovanović-Stojanov, Sofija; Martinović, Vesna; Ivanović-Matić, Svetlana; Stevanović, Jelena; Poznanović, Goran; Grigorov, Ilijana

    2017-11-01

    The progression of oxidative stress, resulting cell damage, and cell death underlies the etiology of liver damage/dysfunction as a complication of diabetes. High-mobility group box 1 (HMGB1) protein, a chromatin-binding nuclear protein and damage-associated molecular pattern molecule, is integral to oxidative stress and signaling pathways regulating cell death and cell survival. We previously found that in streptozotocin (STZ)-induced diabetic rats, reduction of oxidative stress after melatonin administration lowered necrotic cell death and increased expression of HMGB1 and hepatocellular damage. In the present study, we examined whether alleviation of diabetes-attendant oxidative stress and ensuing change in HMGB1 expression influence the dynamic equilibrium between apoptosis/autophagy and liver damage. We observed that elevated HMGB1 protein levels in diabetic rat liver accompanied increased interactions of HMGB1 with TLR4 and RAGE, and activation of the intrinsic apoptotic pathway and Beclin 1-dependent autophagy. The absence of p62 degradation in diabetic rat liver pointed to defective autophagy which was responsible for lower autophagosome/autophagolysosome formation and an increased apoptosis/autophagy ratio. Compared to diabetic rats, in melatonin-treated diabetic rats, the structure of liver cells was preserved, HMGB1/TLR4 interaction and downstream apoptotic signaling were significantly reduced, HMGB1/Beclin 1 colocalization and interactions were augmented and Beclin 1-mediated autophagy, mithophagy in particular, were increased. We concluded that in mild oxidative stress, HMGB1 is cytoprotective, whereas in intense oxidative stress, HMGB1 actions promote cell death and liver damage. Since reduced HMGB1 binds to RAGE but not to TLR4, redox modification of HMGB1 as a mechanism regulating the cross-talk between apoptosis and autophagy in diabetes is discussed.

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

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    Yongke Lu

    2015-10-01

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

  16. Non-canonical autophagy: an exception or an underestimated form of autophagy?

    Science.gov (United States)

    Scarlatti, Francesca; Maffei, Roberta; Beau, Isabelle; Ghidoni, Riccardo; Codogno, Patrice

    2008-11-01

    Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.

  17. Decreased expression of Beclin 1 correlates closely with Bcl-xL expression and poor prognosis of ovarian carcinoma.

    Directory of Open Access Journals (Sweden)

    Huan-Xin Lin

    Full Text Available It has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.In the present study, the methods of Western blotting and immunohistochemistry (IHC were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (P<0.05. In univariate survival analysis, a highly significant association between low-expressed Beclin 1 and shortened patient survival was evaluated in ovarian carcinoma patients (P<0.01, and Beclin 1 expression was an independent prognostic factor as evidenced by multivariate analysis (P = 0.013. In addition, decreased expression of Beclin 1 was inversely correlated with altered expression of Bcl-xL in ovarian carcinoma cohort, and combined analysis further showed that the low Beclin 1/high Bcl-xL group had the lowest survival rate.Our findings suggest that Beclin 1 expression, as examined by IHC, could be served as an additional tool in identifying ovarian carcinoma patients at risk of tumor progression, and predicting patient survival in ovarian carcinomas with increased expression of Bcl-xL.

  18. Inhibition of autophagy by TAB2 and TAB3.

    Science.gov (United States)

    Criollo, Alfredo; Niso-Santano, Mireia; Malik, Shoaib Ahmad; Michaud, Mickael; Morselli, Eugenia; Mariño, Guillermo; Lachkar, Sylvie; Arkhipenko, Alexander V; Harper, Francis; Pierron, Gérard; Rain, Jean-Christophe; Ninomiya-Tsuji, Jun; Fuentes, José M; Lavandero, Sergio; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Kroemer, Guido

    2011-11-11

    Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

  19. Expression of beclin 1 in primary salivary adenoid cystic carcinoma and its relation to Bcl-2 and p53 and prognosis

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    Jiang, L.C.; Huang, S.Y.; Zhang, D.S.; Zhang, S.H.; Li, W.G.; Zheng, P.H.; Chen, Z.W. [Shandong Provincial Hospital Affiliated to Shandong University, Department of Oral and Maxillofacial Surgery, Jinan, China, Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan (China)

    2014-03-03

    Beclin 1 plays a critical role in autophagy and functions as a haploinsufficient tumor suppressor. The expression and prognostic significance of beclin 1 in head and neck adenoid cystic carcinoma (ACC) are largely unexplored. Therefore, we investigated the expression of beclin 1, Bcl-2, and p53 in head and neck ACC tissue. Tissue samples from 35 cases (15 females, 20 males) of head and neck ACC were utilized for immunohistochemistry. Beclin 1 expression was observed in 32 cases (91.4%) and considered to be high in 15 cases (42.9%) and low in 20 cases (57.1%). Beclin 1 expression was significantly correlated with a histological growth pattern (P=0.046) and histological grade (P=0.037). Beclin 1 expression was inversely correlated with Bcl-2 expression (P=0.013) and significantly associated with overall survival (P=0.006). Bcl-2 and p53 expression were observed in 21 cases (60.0%) and 16 cases (45.7%). Bcl-2 expression was significantly correlated with perineural invasion (P=0.041) and not associated with overall survival (P=0.053). p53 expression was directly correlated with beclin 1 expression (P=0.044). Our results indicated that beclin 1 may be a novel, promising prognostic factor for clinical outcome in head and neck ACC patients and may play a part in the development of head and neck ACC by interacting with Bcl-2 and p53.

  20. 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.

  1. Functional Analysis of the Beclin-1 Tumor Suppressor Interaction with hVps34 (Type-III P13’-kinase) in Breast Cancer Cells

    Science.gov (United States)

    2008-06-01

    Methods for monitoring autophagy. Int.J Biochem.Cell Biol., 36: 2491-2502, 2004. 13. Scarlatti , F., Bauvy, C., Ventruti, A., Sala, G., Cluzeaud, F...J.Biol.Chem., 279: 18384-18391, 2004. 14. Scarlatti , F., Maffei, R., Beau, I., Codogno, P., and Ghidoni, R. Role of non-canonical Beclin 1- independent

  2. Induction of cytoprotective autophagy in PC-12 cells by cadmium

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    Wang, Qiwen; Zhu, Jiaqiao; Zhang, Kangbao; Jiang, Chenyang; Wang, Yi; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong; Liu, Zongping

    2013-01-01

    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity

  3. Expression and clinical implication of Beclin1, HMGB1, p62, survivin, BRCA1 and ERCC1 in epithelial ovarian tumor tissues.

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    Ju, L-L; Zhao, C Y; Ye, K-F; Yang, H; Zhang, J

    2016-05-01

    The aim of the present study is to investigate the differential expression of Beclin1, HMGB1, p62, survivin, ERCC1 and BRCA1 protein in epithelial ovarian cancer (EOC) and to evaluate the relationship between autophagy and platinum resistance of EOC patients during platinum-based chemotherapy with the protein expression. Expression of Beclin1, HMGB1, p62, survivin, ERCC1 and BRCA1 were detected with immunohistochemistry in 60 patients, including 39 with epithelial ovarian cancer (EOC), 13 benign epithelial ovarian tumor tissue (BET) and 8 borderline ovarian tumor tissue. Beclin, p62 and ERCC1 expression was significantly higher in the EOC than the BET (p0.05). BRCA1 expression was lower in EOC than BET (pepithelial ovarian cancer.

  4. Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

    Science.gov (United States)

    Zhen, Zijun; Yang, Kaibin; Ye, Litong; You, Zhiyao; Chen, Rirong; Liu, Ying; He, Youjian

    2017-07-01

    Paclitaxel is not as effective for neuroblastoma as most of the front-line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel-associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy-associated proteins were assessed by western blot. Autophagy was induced and the autophagy-associated proteins LC3-I, LC3-II, Beclin 1, and thioredoxin-related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1-mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel-induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel-induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  5. Role of ROS-mediated autophagy in radiation-induced bystander effect of hepatoma cells.

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    Wang, Xiangdong; Zhang, Jianghong; Fu, Jiamei; Wang, Juan; Ye, Shuang; Liu, Weili; Shao, Chunlin

    2015-05-01

    Autophagy plays a crucial role in cellular response to ionizing radiation, but it is unclear whether autophagy can modulate radiation-induced bystander effect (RIBE). Here, we investigated the relationship between bystander damage and autophagy in human hepatoma cells of HepG2. HepG2 cells were treated with conditioned medium (CM) collected from 3 Gy γ-rays irradiated hepatoma HepG2 cells for 4, 12, or 24 h, followed by the measurement of micronuclei (MN), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 in the bystander HepG2 cells. In some experiments, the bystander HepG2 cells were respectively transfected with LC3 small interfering RNA (siRNA), Beclin-1 siRNA or treated with 1% dimethyl sulfoxide (DMSO). Additional MN and mitochondrial dysfunction coupled with ROS were induced in the bystander cells. The expressions of protein markers of autophagy, LC3-II/LC3-I and Beclin-1, increased in the bystander cells. The inductions of bystander MN and overexpressions of LC3 and Beclin-1 were significantly diminished by DMSO. However, when the bystander cells were transfected with LC3 siRNA or Beclin-1 siRNA, the yield of bystander MN was significantly enhanced. The elevated ROS have bi-functions in balancing the bystander effects. One is to cause MN and the other is to induce protective autophagy.

  6. Functional Analysis of the Beclin-1 Tumor Suppressor Interaction With hVps34 (Type-III P13-Kinase) in Breast Cancer Cells

    Science.gov (United States)

    2006-06-01

    function of Vps34 in autophagy by some mechanism other than directing its membrane targeting. This leaves us with the alternative possibility that Beclin...with annexin-V. Annexin-positive cells were counted using a Guava personal cytometer. Cells treated overnight with TNF- (white) served as a positive...stained with annexin-V and 7-amino-actinomycin D as described in the protocol for the Guava NexinTM kit ( Guava Technologies). Staining was quantified

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

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    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.

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

    International Nuclear Information System (INIS)

    Dong, Chenglong; Zheng, Haining; Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng; Ding, Dafa; Lu, Yibing

    2015-01-01

    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

  9. The dual role of autophagy under hypoxia-involvement of interaction between autophagy and apoptosis.

    Science.gov (United States)

    Li, Mengmeng; Tan, Jin; Miao, Yuyang; Lei, Ping; Zhang, Qiang

    2015-06-01

    Hypoxia is one of severe cellular stress and it is well known to be associated with a worse outcome since a lack of oxygen accelerates the induction of apoptosis. Autophagy, an important and evolutionarily conserved mechanism for maintaining cellular homeostasis, is closely related to the apoptosis caused by hypoxia. Generally autophagy blocks the induction of apoptosis and inhibits the activation of apoptosis-associated caspase which could reduce cellular injury. However, in special cases, autophagy or autophagy-relevant proteins may help to induce apoptosis, which could aggravate cell damage under hypoxia condition. In addition, the activation of apoptosis-related proteins-caspase can also degrade autophagy-related proteins, such as Atg3, Atg4, Beclin1 protein, inhibiting autophagy. Although the relationship between autophagy and apoptosis has been known for rather complex for more than a decade, the underlying regulatory mechanisms have not been clearly understood. This short review discusses and summarizes the dual role of autophagy and the interaction and molecular regulatory mechanisms between autophagy and apoptosis under hypoxia.

  10. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

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    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  11. Hypercholesterolemia downregulates autophagy in the rat heart.

    Science.gov (United States)

    Giricz, Zoltán; Koncsos, Gábor; Rajtík, Tomáš; Varga, Zoltán V; Baranyai, Tamás; Csonka, Csaba; Szobi, Adrián; Adameová, Adriana; Gottlieb, Roberta A; Ferdinandy, Péter

    2017-03-23

    We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. Male Wistar rats were fed either normal chow (NORM; n = 9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n = 9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09 mmol/L vs. 2.89 mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the

  12. 18α-Glycyrrhetinic acid lethality for neuroblastoma cells via de-regulating the Beclin-1/Bcl-2 complex and inducing apoptosis.

    Science.gov (United States)

    Rahman, Md Ataur; Bishayee, Kausik; Habib, Khadija; Sadra, Ali; Huh, Sung-Oh

    2016-10-01

    18α-Glycyrrhetinic acid (18-GA) is a known gap-junction inhibitor with demonstrated anticancer effects. However, the different modes of cell cytotoxicity for 18-GA remain to be characterized. In this study, 18-GA reduced the expression of cell-cell interaction proteins (N- and VE-cadherin), and led to a dose-dependent increase in cytotoxicity of the neuroblastoma cells tested, but was less toxic toward actively dividing human embryonic kidney cells. We found that 18-GA could induce both autophagy and apoptosis. 18-GA mediated autophagy was due to accumulation of Atg5, Atg7 and LC3II and degradation of p62. Individual siRNAs against Atg5 and Atg7 prevented autophagy and resulted in a further loss of viability with 18-GA. In addition, combination of 18-GA with autophagy inhibitor chloroquine produced a more significant cell death. This implied a pro-survival function for autophagy induction with 18-GA. 18-GA also led to the destabilization of Bcl-2/Beclin-1 interaction and cleavage of Beclin-1, a protein known to play role in apoptosis and autophagy induction. Treatment of cells by a pan-caspase inhibitor or a caspase-3 siRNA prevented a large portion of 18-GA mediated cytotoxicity, demonstrating that caspase-dependent apoptosis induction was responsible for most of the observed cytotoxicity. In terms of signaling, 18-GA led to reduced phosphorylation of all three classes of MAP kinases. Taken together, 18-GA or its pathways may lead to more effective, targeted therapeutics against neuroblastoma. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Antioxidant Supplement Inhibits Skeletal Muscle Constitutive Autophagy rather than Fasting-Induced Autophagy in Mice

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    Zhengtang Qi

    2014-01-01

    Full Text Available In this study, we tested the hypothesis that NAC administration leads to reduced oxidative stress and thus to decreased expression of autophagy markers in young mice. Our results reveal that NAC administration results in reduced muscle mRNA levels of several autophagy markers, including Beclin-1, Atg7, LC3, Atg9, and LAMP2. However, NAC supplement fails to block the activation of skeletal muscle autophagy in response to fasting, because fasting significantly increases the mRNA level of several autophagy markers and LC3 lipidation. We further examined the effects of NAC administration on mitochondrial antioxidant capacity in fed and 24-hour fasted mice. Our results clearly show that NAC administration depresses the expression of manganese superoxide dismutase (MnSOD and TP53-induced glycolysis and apoptosis regulator (TIGAR, both of which play a predominant antioxidant role in mitochondria by reducing ROS level. In addition, we found no beneficial effect of NAC supplement on muscle mass but it can protect from muscle loss in response to fasting. Collectively, our findings indicate that ROS is required for skeletal muscle constitutive autophagy, rather than starvation-induced autophagy, and that antioxidant NAC inhibits constitutive autophagy by the regulation of mitochondrial ROS production and antioxidant capacity.

  14. Autophagy regulates the stemness of cervical cancer stem cells

    Directory of Open Access Journals (Sweden)

    Yang Y

    2017-06-01

    Full Text Available Yi Yang,1,2 Li Yu,1 Jin Li,1 Ya Hong Yuan,1 Xiao Li Wang,1 Shi Rong Yan,1 Dong Sheng Li,1 Yan Ding1 1Hubei Key Laboratory of Embryonic Stem Cell Research, 2Reproductive Center, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China Abstract: Cancer stem cells (CSCs are a rare population of multipotent cells with the capacity to self-renew. It has been reported that there are CSCs in cervical cancer cells. Pluripotency-associated (PA transcription factors such as Oct4, Sox2, Nanog and CD44 have been used to isolate CSCs subpopulations. In this study, we showed that autophagy plays an important role in the biological behavior of cervical cancer cells. The expression of the autophagy protein Beclin 1 and LC3B was higher in tumorspheres established from human cervical cancers cell lines (and CaSki than in the parental adherent cells. It was also observed that the basal and starvation-induced autophagy flux was higher in tumorspheres than in the bulk population. Autophagy could regulate the expression level of PA proteins in cervical CSCs. In addition, CRISPR/Cas 9-mediated Beclin 1 knockout enhanced the malignancy of HeLa cells, leading to accumulation of PA proteins and promoted tumorsphere formation. Our findings suggest that autophagy modulates homeostasis of PA proteins, and Beclin 1 is critical for CSC maintenance and tumor development in nude mice. This demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance. Keywords: cervical cancer, autophagy, cancer stem cell, LC3, Oct4

  15. Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling.

    Science.gov (United States)

    Zhang, Qiuxia; Na, Quan; Song, Weiwei

    2017-10-01

    Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling. © 2017 Japan Society of Obstetrics and Gynecology.

  16. Cantharidin Inhibits the Growth of Triple-Negative Breast Cancer Cells by Suppressing Autophagy and Inducing Apoptosis in Vitro and in Vivo

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    Hong-chang Li

    2017-10-01

    Full Text Available Background/Aims: Cantharidin, a type of terpenoid secreted by the blister beetle Mylabris phalerata (Pallas, has attracted great attention in cancer therapy because of its potential anti-cancer activities. Here, we report the effects on apoptosis and autophagy in human triple-negative breast cancer (TNBC cell lines after treatment with cantharidin and attempt to elucidate the underlying mechanisms. Methods: MDA-MB-231 and MDA-MB-468 cells were treated with cantharidin and cell proliferation was examined using CCK-8 and clone formation assays. The expression of apoptosis- and autophagy-associated proteins was detected by western blotting. Cells were infected with lentivirus carrying the Beclin-1 gene, and MDA-MB-231-beclin1 (MB231-Bec and MDA-MB-468-beclin-1(MB468-Bec cells stably expressing Beclin-1 were established. Autophagic vacuoles in cells were observed with LC3 staining using fluorescence microscopy, and apoptotic cells were detected via flow cytometry. Tumor growth was assessed by subcutaneous inoculation of TNBC cells into BALB/c nude mice. Results: Cantharidin inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells, and induced cell apoptosis. Cantharidin additionally inhibited the conversion of LC3 I to LC3 II and autophagosome formation by suppressing the expression of Beclin-1. Furthermore, overexpression of Beclin-1 in TNBC cells attenuated the cytotoxicity of cantharidin. In vivo, cantharidin inhibited the growth of MDA-MB-231 and MDA-MB-468 xenografts in nude mice by suppressing autophagy and inducing apoptosis, and Beclin-1 overexpression in TNBC cells reduced the efficacy of cantharidin. Conclusions: Cantharidin inhibits autophagy by suppressing Beclin-1 expression and inducing apoptosis of TNBC cells in vitro and in vivo, thereby representing a potential strategy for the treatment of TNBC.

  17. Autophagy response in the liver of pigeon exposed to avermectin.

    Science.gov (United States)

    Wang, Xian-Song; Liu, Ci; Khoso, Pervez Ahmed; Zheng, Weijia; Li, Ming; Li, Shu

    2017-05-01

    Pesticide residues are an important aspect of environmental pollution. Environmental avermectin residues have produced adverse effects in organisms. Many pesticides exert their toxic effects via the mechanism of autophagy. The purpose of this study was to examine the changes in autophagy levels and in autophagy-related genes, including LC3, Beclin 1, Dynein, ATG5, TORC1, and TORC2, resulting from exposure to subchronic levels of AVM in liver tissue in the king pigeon model. We observed abundant autophagic vacuoles with extensively degraded organelles, autophagosomal vacuoles, secondary lysosomes, and double-membrane structures in the liver. The expression levels of the autophagy-related genes LC3-I, LC3-II, Beclin 1, ATG5, and Dynein were up-regulated; however, TORC1 and TORC2 expression levels were down-regulated. These changes occurred in a concentration-dependent manner after AVM exposure for 30, 60, and 90 days in pigeons. Taken together, these results suggested that AVM increased the autophagic flux and that upregulation of autophagy might be closely related to the hepatotoxicity of AVM in birds.

  18. Knockdown of autophagy enhances innate immune response in hepatitis C virus infected hepatocytes

    Science.gov (United States)

    Shrivastava, Shubham; Raychoudhuri, Amit; Steele, Robert; Ray, Ranjit; Ray, Ratna B.

    2010-01-01

    The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of innate immune system against viral infection. In the present study, we have shown that knockdown of autophagy related protein Beclin1 or ATG7 in immortalized human hepatocytes (IHH) inhibited HCV growth. Beclin1 or ATG7 knockdown IHH when infected with HCV exhibited an increased expression of IFN-β, OAS-1, IFN-α and IFI27 mRNAs of the interferon signaling pathways as compared to infection of control IHH. Subsequent study demonstrated that HCV infection in autophagy impaired IHH displayed caspase activation, PARP cleavage and apoptotic cell death. Conclusion The disruption of autophagy machinery in HCV infected hepatocytes activated IFN signaling pathway, and induced apoptosis. Together, these results suggest that HCV induced autophagy impairs innate immune response. PMID:21274862

  19. Lipopolysaccharide stimulates p62-dependent autophagy-like aggregate clearance in hepatocytes.

    Science.gov (United States)

    Chen, Christine; Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R; Scott, Melanie J

    2014-01-01

    Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NF κ B transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

  20. Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation.

    Science.gov (United States)

    Lavieu, Grégory; Scarlatti, Francesca; Sala, Giusy; Carpentier, Stéphane; Levade, Thierry; Ghidoni, Riccardo; Botti, Joëlle; Codogno, Patrice

    2006-03-31

    The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.

  1. 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.

  2. The interplay between autophagy and ROS in tumorigenesis

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    Kongara, Sameera [Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ (United States); The Cancer Institute of New Jersey, New Brunswick, NJ (United States); Karantza, Vassiliki, E-mail: karantva@umdnj.edu [Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ (United States); The Cancer Institute of New Jersey, New Brunswick, NJ (United States); Division of Medical Oncology, Department of Internal Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ (United States)

    2012-11-21

    Reactive oxygen species (ROS) at physiological levels are important cell signaling molecules. However, aberrantly high ROS are intimately associated with disease and commonly observed in cancer. Mitochondria are primary sources of intracellular ROS, and their maintenance is essential to cellular health. Autophagy, an evolutionarily conserved process whereby cytoplasmic components are delivered to lysosomes for degradation, is responsible for mitochondrial turnover and removal of damaged mitochondria. Impaired autophagy is implicated in many pathological conditions, including neurological disorders, inflammatory bowel disease, diabetes, aging, and cancer. The first reports connecting autophagy to cancer showed that allelic loss of the essential autophagy gene BECLIN1 (BECN1) is prevalent in human breast, ovarian, and prostate cancers and that Becn1{sup +/-} mice develop mammary gland hyperplasias, lymphomas, lung and liver tumors. Subsequent studies demonstrated that Atg5{sup -/-} and Atg7{sup -/-} livers give rise to adenomas, Atg4C{sup -/-} mice are susceptible to chemical carcinogenesis, and Bif1{sup -/-} mice are prone to spontaneous tumors, indicating that autophagy defects promote tumorigenesis. Due to defective mitophagy, autophagy-deficient cells accumulate damaged mitochondria and deregulated ROS levels, which likely contribute to their tumor-initiating capacity. However, the role of autophagy in tumorigenesis is complex, as more recent work also revealed tumor dependence on autophagy: autophagy-competent mutant-Ras-expressing cells form tumors more efficiently than their autophagy-deficient counterparts; similarly, FIP200 deficiency suppresses PyMT-driven mammary tumorigenesis. These latter findings are attributed to the fact that tumors driven by powerful oncogenes have high metabolic demands catered to by autophagy. In this review, we discuss the relationship between ROS and autophagy and summarize our current knowledge on their functional interactions

  3. The interplay between autophagy and ROS in tumorigenesis

    International Nuclear Information System (INIS)

    Kongara, Sameera; Karantza, Vassiliki

    2012-01-01

    Reactive oxygen species (ROS) at physiological levels are important cell signaling molecules. However, aberrantly high ROS are intimately associated with disease and commonly observed in cancer. Mitochondria are primary sources of intracellular ROS, and their maintenance is essential to cellular health. Autophagy, an evolutionarily conserved process whereby cytoplasmic components are delivered to lysosomes for degradation, is responsible for mitochondrial turnover and removal of damaged mitochondria. Impaired autophagy is implicated in many pathological conditions, including neurological disorders, inflammatory bowel disease, diabetes, aging, and cancer. The first reports connecting autophagy to cancer showed that allelic loss of the essential autophagy gene BECLIN1 (BECN1) is prevalent in human breast, ovarian, and prostate cancers and that Becn1 +/- mice develop mammary gland hyperplasias, lymphomas, lung and liver tumors. Subsequent studies demonstrated that Atg5 -/- and Atg7 -/- livers give rise to adenomas, Atg4C -/- mice are susceptible to chemical carcinogenesis, and Bif1 -/- mice are prone to spontaneous tumors, indicating that autophagy defects promote tumorigenesis. Due to defective mitophagy, autophagy-deficient cells accumulate damaged mitochondria and deregulated ROS levels, which likely contribute to their tumor-initiating capacity. However, the role of autophagy in tumorigenesis is complex, as more recent work also revealed tumor dependence on autophagy: autophagy-competent mutant-Ras-expressing cells form tumors more efficiently than their autophagy-deficient counterparts; similarly, FIP200 deficiency suppresses PyMT-driven mammary tumorigenesis. These latter findings are attributed to the fact that tumors driven by powerful oncogenes have high metabolic demands catered to by autophagy. In this review, we discuss the relationship between ROS and autophagy and summarize our current knowledge on their functional interactions in tumorigenesis.

  4. Anti- and pro-tumor functions of autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Galluzzi, Lorenzo; Kepp, Oliver; Vicencio, José-Miguel; Criollo, Alfredo; Maiuri, Maria Chiara; Kroemer, Guido

    2009-09-01

    Autophagy constitutes one of the major responses to stress in eukaryotic cells, and is regulated by a complex network of signaling cascades. Not surprisingly, autophagy is implicated in multiple pathological processes, including infection by pathogens, inflammatory bowel disease, neurodegeneration and cancer. Both oncogenesis and tumor survival are influenced by perturbations of the molecular machinery that controls autophagy. Numerous oncoproteins, including phosphatidylinositol 3-kinase, Akt1 and anti-apoptotic members of the Bcl-2 family suppress autophagy. Conversely, several tumor suppressor proteins (e.g., Atg4c; beclin 1; Bif-1; BH3-only proteins; death-associated protein kinase 1; LKB1/STK11; PTEN; UVRAG) promote the autophagic pathway. This does not entirely apply to p53, one of the most important tumor suppressor proteins, which regulates autophagy in an ambiguous fashion, depending on its subcellular localization. Irrespective of the controversial role of p53, basal levels of autophagy appear to inhibit tumor development. On the contrary, chemotherapy- and metabolic stress-induced activation of the autophagic pathway reportedly contribute to the survival of formed tumors, thereby favoring resistance. In this context, autophagy inhibition would represent a major therapeutic target for chemosensitization. Here, we will review the current knowledge on the dual role of autophagy as an anti- and pro-tumor mechanism.

  5. Nutrient-deprivation autophagy factor-1 (NAF-1: biochemical properties of a novel cellular target for anti-diabetic drugs.

    Directory of Open Access Journals (Sweden)

    Sagi Tamir

    Full Text Available Nutrient-deprivation autophagy factor-1 (NAF-1 (synonyms: Cisd2, Eris, Miner1, and Noxp70 is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER and mitochondrial outer membrane. It was implicated in human pathology (Wolfram Syndrome 2 and in BCL-2 mediated antagonization of Beclin 1-dependent autophagy and depression of ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties of its 2Fe-2S cluster and sensitivity of those properties to small molecules. The structure of the soluble domain of NAF-1 shows that it forms a homodimer with each protomer containing a [2Fe-2S] cluster bound by 3 Cys and one His. NAF-1 has shown the unusual abilities to transfer its 2Fe-2S cluster to an apo-acceptor protein (followed in vitro by spectrophotometry and by native PAGE electrophoresis and to transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron fluorescent sensors targeted to mitochondria. Importantly, the drug pioglitazone abrogates NAF-1's ability to transfer the cluster to acceptor proteins and iron to mitochondria. Similar effects were found for the anti-diabetes and longevity-promoting antioxidant resveratrol. These results reveal NAF-1 as a previously unidentified cell target of anti-diabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster.

  6. Homocysteine Aggravates Cortical Neural Cell Injury through Neuronal Autophagy Overactivation following Rat Cerebral Ischemia-Reperfusion

    Directory of Open Access Journals (Sweden)

    Yaqian Zhao

    2016-07-01

    Full Text Available Elevated homocysteine (Hcy levels have been reported to be involved in neurotoxicity after ischemic stroke. However, the underlying mechanisms remain incompletely understood to date. In the current study, we hypothesized that neuronal autophagy activation may be involved in the toxic effect of Hcy on cortical neurons following cerebral ischemia. Brain cell injury was determined by hematoxylin-eosin (HE staining and TdT-mediated dUTP Nick-End Labeling (TUNEL staining. The level and localization of autophagy were detected by transmission electron microscopy, western blot and immunofluorescence double labeling. The oxidative DNA damage was revealed by immunofluorescence of 8-Hydroxy-2′-deoxyguanosine (8-OHdG. Hcy treatment aggravated neuronal cell death, significantly increased the formation of autophagosomes and the expression of LC3B and Beclin-1 in the brain cortex after middle cerebral artery occlusion-reperfusion (MCAO. Immunofluorescence analysis of LC3B and Beclin-1 distribution indicated that their expression occurred mainly in neurons (NeuN-positive and hardly in astrocytes (GFAP-positive. 8-OHdG expression was also increased in the ischemic cortex of Hcy-treated animals. Conversely, LC3B and Beclin-1 overexpression and autophagosome accumulation caused by Hcy were partially blocked by the autophagy inhibitor 3-methyladenine (3-MA. Hcy administration enhanced neuronal autophagy, which contributes to cell death following cerebral ischemia. The oxidative damage-mediated autophagy may be a molecular mechanism underlying neuronal cell toxicity of elevated Hcy level.

  7. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

    Science.gov (United States)

    Scarlatti, Francesca; Bauvy, Chantal; Ventruti, Annamaria; Sala, Giusy; Cluzeaud, Françoise; Vandewalle, Alain; Ghidoni, Riccardo; Codogno, Patrice

    2004-04-30

    The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

  8. Breviscapine confers a neuroprotective efficacy against transient focal cerebral ischemia by attenuating neuronal and astrocytic autophagy in the penumbra.

    Science.gov (United States)

    Pengyue, Zhang; Tao, Guo; Hongyun, He; Liqiang, Yang; Yihao, Deng

    2017-06-01

    Breviscapine is a flavonoid derived from a traditional Chinese herb Erigerin breviscapus (Vant.) Hand-Mazz, and has been extensively used in clinical treatment for cerebral stroke in China, but the underlying pharmacological mechanisms are still unclear. In present study, we investigated whether breviscapine could confer a neuroprotection against cerebral ischemia injury by targeting autophagy mechanisms. A cerebral stroke model in Sprague-Dawley rats was prepared by middle cerebral artery occlusion (MCAO), rats were then randomly divided into 5 groups: MCAO+Bre group, rats were treated with breviscapine; MCAO+Tat-Beclin-1 group, animals were administrated with specific autophagy inducer Tat-Beclin-1; MCAO+Bre+Tat-Beclin-1 group, rats were treated with both breviscapine and Tat-Beclin-1, MCAO+saline group, rats received the same volume of physiological saline, and Sham surgery group. The autophagy levels in infarct penumbra were evaluated by western blotting, real-time PCR and immunofluorescence 7days after the insult. Meanwhile, infarct volume, brain water content and neurological deficit score were assessed. The results illustrated that the infarct volume, brain water content and neurofunctional deficiency were significantly reduced by 7days of breviscapine treatment in MCAO+Bre group, compared with those in MCAO+saline group. Meanwhile, the western blotting, quantitative PCR and immunofluorescence showed that the autophagy in both neurons and astrocytes at the penumbra were markedly attenuated by breviscapine admininstration. Moreover, these pharmacological effects of breviscapine could be counteracted by autophagy inducer Tat-Beclin-1. Our study suggests that breviscapine can provide a neuroprotection against transient focal cerebral ischemia, and this biological function is associated with attenuating autophagy in both neurons and astrocytes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. WW domain of BAG3 is required for the induction of autophagy in glioma cells.

    Science.gov (United States)

    Merabova, Nana; Sariyer, Ilker Kudret; Saribas, A Sami; Knezevic, Tijana; Gordon, Jennifer; Turco, M Caterina; Rosati, Alessandra; Weaver, Michael; Landry, Jacques; Khalili, Kamel

    2015-04-01

    Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associates with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. © 2014 Wiley Periodicals, Inc.

  10. Increased autophagy in placentas of intrauterine growth-restricted pregnancies.

    Directory of Open Access Journals (Sweden)

    Tai-Ho Hung

    Full Text Available Unexplained intrauterine growth restriction (IUGR may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1 to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2 to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3 to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts.Compared with normal pregnant women, women with IUGR had higher placental levels of autophagy-related proteins LC3B-II, beclin-1, and damage-regulated autophagy modulator (DRAM, with increased p53 and caspase-cleaved cytokeratin 18 (M30. Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen in the presence or absence of nutlin-3 (a p53 activity stimulator had higher levels of LC3B-II, DRAM, and M30 proteins and increased Bax mRNA expression compared with controls cultured under standard conditions. In contrast, administration of pifithrin-α (a p53 activity inhibitor during hypoxia resulted in protein levels that were similar to those of the control groups. Moreover, cytotrophoblasts transfected with LC3B, beclin-1, or DRAM siRNA had higher levels of M30 compared with the controls under hypoxia. However, transfection with Bcl-2 or Bax siRNA did not cause any significant change in the levels of LC3B-II in hypoxic cytotrophoblasts.Together, these results suggest that there is a crosstalk between autophagy and apoptosis in IUGR and that p53 plays a pivotal and complex role in regulating trophoblast cell turnover in response to hypoxic stress.

  11. 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...... as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR...

  12. The inositol trisphosphate receptor in the control of autophagy.

    Science.gov (United States)

    Criollo, Alfredo; Vicencio, José Miguel; Tasdemir, Ezgi; Maiuri, M Chiara; Lavandero, Sergio; Kroemer, Guido

    2007-01-01

    The second messenger myo-inositol-1,4,5-trisphosphate (IP(3)) acts on the IP(3) receptor (IP(3)R), an IP(3)-activated Ca(2+) channel of the endoplasmic reticulum (ER). The IP(3)R agonist IP(3) inhibits starvation-induced autophagy. The IP(3)R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-XL, two proteins that physically interact with IP(3)R. Autophagy can also be induced by depletion of the IP(3)R by small interfering RNAs. Autophagy induction by IP(3)R blockade cannot be explained by changes in steady state levels of Ca(2+) in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP(3)R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

  13. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    Science.gov (United States)

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. 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.

  15. Myocardial Autophagy after Severe Burn in Rats

    Science.gov (United States)

    Zhang, Qiong; Shi, Xiao-hua; Huang, Yue-sheng

    2012-01-01

    Background Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn. Methods Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function. Results Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function. Conclusion Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction. PMID:22768082

  16. 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.

  17. [Astragalus polysaccharide may increase sensitivity of cervical cancer HeLa cells to cisplatin by regulating cell autophagy].

    Science.gov (United States)

    Zhai, Qiu-Li; Hu, Xiang-Dan; Xiao, Jing; Yu, Dong-Qing

    2018-02-01

    This study aimed to investigate the possible sensitivity of Astragalus polysaccharides, in order to improve the chemosensitivity of cervical cancer HeLa cells to cisplatin by regulating the cell autophagy, and explore its possible mechanism. In this study, HeLa cells were divided into control group, cisplatin group, Astragalus polysaccharide group, and Astragalus polysaccharide combined with cisplatin group. MTT assay was used to detect the proliferation of cervical cancer HeLa cells. Flow cytometry was used to detect the apoptosis and cycle of HeLa cells in each experimental group. RT-PCR was used to detect the mRNA expression of autophagy-related proteins beclin1, LC3Ⅱ and p62. The expression levels of autophagy-related proteins beclin1, LC3Ⅱ, LC3Ⅰ and p62 were detected by WB method. MTT results showed that compared with the control group, the proliferation of HeLa cells was significantly inhibited in each administration group( P HeLa cells was significantly increased( P HeLa cells to cisplatin by regulating the cell autophagy. Its possible mechanism of action is correlated with the up-regulation of autophagy-related proteins beclin1, the promote the conversion from LC3Ⅰ to LC3Ⅱ, the down-regulation of labeled protein p62, and the enhancement of HeLa cell autophagic activity, thereby increasing the sensitivity of HeLa cells to cisplatin chemotherapy. Copyright© by the Chinese Pharmaceutical Association.

  18. Targeting autophagy in cancer management – strategies and developments

    International Nuclear Information System (INIS)

    Ozpolat, Bulent; Benbrook, Doris M

    2015-01-01

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

  19. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

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    Chang, Cheng-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan (China); Kuan, Yu-Hsiang [Department of Pharmacology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pharmacy, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Ou, Yen-Chuan; Li, Jian-Ri [Division of Urology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Wu, Chih-Cheng [Department of Anesthesiology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Department of Financial and Computational Mathematics, Providence University, Taichung 433, Taiwan (China); Pan, Pin-Ho [Department of Pediatrics, Tungs’ Taichung MetroHarbor Hospital, Taichung 435, Taiwan (China); Chen, Wen-Ying [Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Huang, Hsuan-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Chen, Chun-Jung, E-mail: cjchen@vghtc.gov.tw [Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan (China); Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Center for General Education, Tunghai University, Taichung 407, Taiwan (China); Department of Nursing, HungKuang University, Taichung 433, Taiwan (China)

    2014-09-10

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK.

  20. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    International Nuclear Information System (INIS)

    Chang, Cheng-Yi; Kuan, Yu-Hsiang; Ou, Yen-Chuan; Li, Jian-Ri; Wu, Chih-Cheng; Pan, Pin-Ho; Chen, Wen-Ying; Huang, Hsuan-Yi; Chen, Chun-Jung

    2014-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK

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

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    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.

  2. Role of autophagy and its molecular mechanisms in mice intestinal tract after severe burn.

    Science.gov (United States)

    Zhang, Duan Y; Qiu, Wei; Jin, PeiS; Wang, Peng; Sun, Yong

    2017-10-01

    Severe burn can lead to hypoxia/ischemia of intestinal mucosa. Autophagy is the process of intracellular degradation, which is essential for cell survival under stresses, such as hypoxia/ischemia and nutrient deprivation. The present study was designed to investigate whether there were changes in intestinal autophagy after severe burn in mice and further to explore the effect and molecular mechanisms of autophagy on intestinal injury. This study includes three experiments. Kunming species mice were subjected to 30% total body surface area third-degree burn. First, we determined protein of LC3 (light chain 3), beclin-1, and cleaved-caspase3 by Western blotting and immunohistochemical (paraffin) staining to investigate whether there were changes in intestinal autophagy after severe burn in mice. Then, changes of the status of enteric damage postburn were measured by observing intestinal mucosa morphology under a magnifier, hematoxylin and eosin staining, enzyme-linked immunosorbent assay, Western blotting under the condition that the intestinal autophagy was respectively activated by rapamycin and inhibited by 3-methyladenine. Finally, protein of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, LC3-II and beclin-1 were assayed, and mice were treated with compound C before burn. The protein of LC3 and beclin-1 were observed at 1 hour postburn and increased to peak-point at 24 hours, reaching the normal level at 96 hours. The cleaved caspase-3 expression increased at 1 hour postburn, but the peak point occurred at 12 hours and had dropped to normal level at 72 hours. In addition, rapamycin enhanced intestinal autophagy and alleviated burn-induced gut damage, while 3-methyladenine showed the against behavior. The AMPK/mTOR pathway which was inhibited decreased the expression of phosphorylated AMPK, LC3-II, and beclin-1, increasing the expression of phosphorylated mTOR. Intestinal autophagy is activated and response to intestinal

  3. 17-AAG enhances the cytotoxicity of flavopiridol in mantle cell lymphoma via autophagy suppression.

    Science.gov (United States)

    Xiao, Y; Guan, J

    2015-01-01

    Flavopiridol, a cyclin-dependent kinase inhibitor (CDKI), shows promising anti-tumor activity in hematologic malignancies. However, Flavopiridol-induced protective autophagy may lead to drug resistance. Here we found that Hsp90 inhibitor 17-AAG can sensitize mantle cell lymphoma (MCL) cells to flavopiridol by suppressing flavopiridol-triggered protective autophagy. The suppressing effect of 17-AAG on autophgy was mediated by Beclin1 degradation and ERK inactivation. Furthermore, 17-AAG enhanced flavopiridol-induced apoptosis and growth suppression in MCL cells. Our study may provide some insights into CDKI -targeted chemotherapies.

  4. Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.

    Science.gov (United States)

    Armstrong, Jane L; Hill, David S; McKee, Christopher S; Hernandez-Tiedra, Sonia; Lorente, Mar; Lopez-Valero, Israel; Eleni Anagnostou, Maria; Babatunde, Fiyinfoluwa; Corazzari, Marco; Redfern, Christopher P F; Velasco, Guillermo; Lovat, Penny E

    2015-06-01

    Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

  5. Galectin-3 and Beclin1/Atg6 genes in human cancers: using cDNA tissue panel, qRT-PCR, and logistic regression model to identify cancer cell biomarkers.

    Directory of Open Access Journals (Sweden)

    Halliday A Idikio

    Full Text Available Cancer biomarkers are sought to support cancer diagnosis, predict cancer patient response to treatment and survival. Identifying reliable biomarkers for predicting cancer treatment response needs understanding of all aspects of cancer cell death and survival. Galectin-3 and Beclin1 are involved in two coordinated pathways of programmed cell death, apoptosis and autophagy and are linked to necroptosis/necrosis. The aim of the study was to quantify galectin-3 and Beclin1 mRNA in human cancer tissue cDNA panels and determine their utility as biomarkers of cancer cell survival.A panel of 96 cDNAs from eight (8 different normal and cancer tissue types were used for quantitative real-time polymerase chain reaction (qRT-PCR using ABI7900HT. Miner2.0, a web-based 4- and 3-parameter logistic regression software was used to derive individual well polymerase chain reaction efficiencies (E and cycle threshold (Ct values. Miner software derived formula was used to calculate mRNA levels and then fold changes. The ratios of cancer to normal tissue levels of galectin-3 and Beclin1 were calculated (using the mean for each tissue type. Relative mRNA expressions for galectin-3 were higher than for Beclin1 in all tissue (normal and cancer types. In cancer tissues, breast, kidney, thyroid and prostate had the highest galectin-3 mRNA levels compared to normal tissues. High levels of Beclin1 mRNA levels were in liver and prostate cancers when compared to normal tissues. Breast, kidney and thyroid cancers had high galectin-3 levels and low Beclin1 levels.Galectin-3 expression patterns in normal and cancer tissues support its reported roles in human cancer. Beclin1 expression pattern supports its roles in cancer cell survival and in treatment response. qRT-PCR analysis method used may enable high throughput studies to generate molecular biomarker sets for diagnosis and predicting cancer treatment response.

  6. Monitoring Autophagy Immunohistochemically and Ultrastructurally during Human Head and Neck Carcinogenesis. Relationship with the DNA Damage Response Pathway †

    Science.gov (United States)

    Havaki, Sophia; Vlachou, Vassiliki; Zampetidis, Christos P.; Selemenakis, Platonas; Kotsinas, Athanassios; Mavrogonatou, Eleni; Rizou, Sophia V.; Kyrodimos, Euthymios; Evangelou, Konstantinos; Kletsas, Dimitris; Giatromanolaki, Alexandra; Gorgoulis, Vassilis G.

    2017-01-01

    Autophagy is a catabolic process that preserves cellular homeostasis. Its exact role during carcinogenesis is not completely defined. Specifically in head and neck cancer, such information from clinical settings that comprise the whole spectrum of human carcinogenesis is very limited. Towards this direction, we examined the in situ status of the autophagy-related factors, Beclin-1, microtubule-associated protein 1 light chain 3, member B (LC3B) and sequestosome 1/p62 (p62) in clinical material covering all histopathological stages of human head and neck carcinogenesis. This material is unique as each panel of lesions is derived from the same patient and moreover we have previously assessed it for the DNA damage response (DDR) activation status. Since Beclin-1, LC3B and p62 reflect the nucleation, elongation and degradation stages of autophagy, respectively, their combined immunohistochemical (IHC) expression profiles could grossly mirror the autophagic flux. This experimental approach was further corroborated by ultrastructural analysis, applying transmission electron microscopy (TEM). The observed Beclin-1/LC3B/p62 IHC patterns, obtained from serial sections analysis, along with TEM findings are suggestive of a declined authophagic activity in preneoplastic lesions that was restored in full blown cancers. Correlating these findings with DDR status in the same pathological stages are indicative of: (i) an antitumor function of autophagy in support to that of DDR, possibly through energy deprivation in preneoplastic stages, thus preventing incipient cancer cells from evolving; and (ii) a tumor-supporting role in the cancerous stage. PMID:28880214

  7. Targeted co-delivery of Beclin 1 siRNA and FTY720 to hepatocellular carcinoma by calcium phosphate nanoparticles for enhanced anticancer efficacy

    Directory of Open Access Journals (Sweden)

    Wu JY

    2018-03-01

    Full Text Available Jun-Yi Wu,1,* Zhong-Xia Wang,1,* Guang Zhang,1 Xian Lu,1 Guang-Hui Qiang,2 Wei Hu,2 An-Lai Ji,3 Jun-Hua Wu,4 Chun-Ping Jiang1 1Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China; 2Department of Hepatobiliary Surgery, Drum Tower Clinical College of Nanjing Medical University, Nanjing, Jiangsu, China; 3Department of General Surgery, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, Jiangsu, China; 4Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing, Jiangsu, China *These authors contributed equally to this work Purpose: FTY720, known as fingolimod, is a new immunosuppressive agent with effective anticancer properties. Although it was recently confirmed that FTY720 inhibits cancer cell proliferation, FTY720 can also induce protective autophagy and reduce cytotoxicity. Blocking autophagy with Beclin 1 siRNA after treatment with FTY720 promotes apoptosis. The objective of this study was to enhance the anticancer effect of FTY720 in hepatocellular carcinoma (HCC by targeted co-delivery of FTY720 and Beclin 1 siRNA using calcium phosphate (CaP nanoparticles (NPs.Materials and methods: First, the siRNA was encapsulated within the CaP core. To form an asymmetric lipid bilayer structure, we then used an anionic lipid for the inner leaflet and a cationic lipid for the outer leaflet; after removing chloroform by rotary evaporation, these lipids were dispersed in a saline solution with FTY720. The NPs were analyzed by transmission electron microscopy, dynamic light scattering and ultraviolet–visible spectrophotometry. Cancer cell viability and cell death were analyzed by MTT assays, fluorescence-activated cell sorting analysis and Western blotting. In addition, the in vivo effects of the NPs were investigated using an athymic nude mouse subcutaneous transplantation tumor model.Results: When the CaP NPs, called

  8. Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.

    Science.gov (United States)

    Fetterman, Jessica L; Holbrook, Monica; Flint, Nir; Feng, Bihua; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Duess, Mai-Ann; Farb, Melissa G; Gokce, Noyan; Shirihai, Orian S; Hamburg, Naomi M; Vita, Joseph A

    2016-04-01

    Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. 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.

  10. Ghrelin improves vascular autophagy in rats with vascular calcification.

    Science.gov (United States)

    Xu, Mingming; Liu, Lin; Song, Chenfang; Chen, Wei; Gui, Shuyan

    2017-06-15

    This study aimed to investigate whether ghrelin ameliorated vascular calcification (VC) through improving autophagy. VC model was induced by nicotine plus vitamin D 3 in rats and β-glycerophosphate in vascular smooth muscle cell (VSMC). Calcium deposition was detected by von Kossa staining or alizarin red S staining. ALP activity was also detected. Western blot was used to assess the protein expression. Ghrelin treatment attenuated the elevation of calcium deposition and ALP activity in VC model both in vivo and in vitro. Interesting, the protein levels of autophagy markers, LC3 and beclin1 were significantly upregulated by ghrelin in VC model. An autophagy inhibitor, 3-methyladenine blocks the ameliorative effect of ghrelin on VC. Furthermore, protein expressions of phosphate-AMPK were increased by ghrelin treatment both in calcified aorta and VSMC. The effect of ghrelin on autophagy induction and VC attenuation was prevented by AMPK inhibitor, compound C. Our results suggested that ghrelin improved autophagy through AMPK activation, which was resulted in VC amelioration. These data maybe throw light on prevention and therapy of VC. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Acid-induced autophagy protects human lung cancer cells from apoptosis by activating ER stress.

    Science.gov (United States)

    Xie, Wen-Yue; Zhou, Xiang-Dong; Li, Qi; Chen, Ling-Xiu; Ran, Dan-Hua

    2015-12-10

    An acidic tumor microenvironment exists widely in solid tumors. However, the detailed mechanism of cell survival under acidic stress remains unclear. The aim of this study is to clarify whether acid-induced autophagy exists and to determine the function and mechanism of autophagy in lung cancer cells. We have found that acute low pH stimulated autophagy by increasing LC3-positive punctate vesicles, increasing LC3 II expression levels and reducing p62 protein levels. Additionally, autophagy was inhibited by the addition of Baf or knockdown of Beclin 1, and cell apoptosis was increased markedly. In mouse tumors, the expression of cleaved caspase3 and p62 was enhanced by oral treatment with sodium bicarbonate, which can raise the intratumoral pH. Furthermore, the protein levels of ER stress markers, including p-PERK, p-eIF2α, CHOP, XBP-1s and GRP78, were also increased in response to acidic pH. The antioxidant NAC, which reduces ROS accumulation, alleviated acid-mediated ER stress and autophagy, and knocking down GRP78 reduced autophagy activation under acidic conditions, which suggests that autophagy was induced by acidic pH through ER stress. Taken together, these results indicate that the acidic microenvironment in non-small cell lung cancer cells promotes autophagy by increasing ROS-ER stress, which serves as a survival adaption in this setting. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Mesenchymal Stem Cells Alleviate LPS-Induced Acute Lung Injury in Mice by MiR-142a-5p-Controlled Pulmonary Endothelial Cell Autophagy

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    Zichao Zhou

    2016-01-01

    Full Text Available Background/Aims: Damages of pulmonary endothelial cells (PECs represent a critical pathological process during acute lung injury (ALI, and precede pulmonary epithelial cell injury, and long-term lung dysfunction. Transplantation of mesenchymal stem cells (MSCs has proven therapeutic effects on ALI, whereas the underlying mechanisms remain ill-defined. Method: We transplanted MSCs in mice and then induced ALI using Lipopolysaccharides (LPS. We analyzed the changes in permeability index and lung histology. Mouse PECs were isolated by flow cytometry based on CD31 expression and then analyzed for autophagy-associated factors LC3 and Beclin-1 by Western blot. Beclin-1 mRNA was determined by RT-qPCR. In vitro, we performed bioinformatics analyses to identify the MSCs-regulated miRNAs that target Beclin-1, and confirmed that the binding was functional by 3'-UTR luciferase reporter assay. Results: We found that MSCs transplantation significantly reduced the severity of LPS-induced ALI in mice. MSCs increased autophagy of PECs to promote PEC survival. MSCs increased Beclin-1 protein but not mRNA. MiR-142a-5p was found to target the 3'-UTR of Beclin-1 mRNA to inhibit its protein translation in PECs. MSCs reduced the levels of miR-142a-5p in PECs from LPS-treated mice. Conclusion: MSCs may alleviate LPS-ALI through downregulation of miR-142a-5p, which allows PECs to increase Beclin-1-mediated cell autophagy.

  13. Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary.

    Science.gov (United States)

    Morais, Roberto D V S; Thomé, Ralph G; Santos, Hélio B; Bazzoli, Nilo; Rizzo, Elizete

    2016-04-01

    In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

    International Nuclear Information System (INIS)

    Ristic, Biljana; Bosnjak, Mihajlo; Arsikin, Katarina; Mircic, Aleksandar; Suzin-Zivkovic, Violeta; Bogdanovic, Andrija; Perovic, Vladimir; Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

    2014-01-01

    We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade.

  15. Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Ristic, Biljana [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Bosnjak, Mihajlo [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Arsikin, Katarina [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Mircic, Aleksandar; Suzin-Zivkovic, Violeta [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Bogdanovic, Andrija [Clinic for Hematology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade (Serbia); Perovic, Vladimir [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Trajkovic, Vladimir, E-mail: vtrajkovic@med.bg.ac.rs [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Harhaji-Trajkovic, Ljubica, E-mail: buajk@yahoo.com [Institute for Biological Research, University of Belgrade, Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade (Serbia)

    2014-08-01

    We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade.

  16. Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae-Sung, E-mail: Jae.Kim@surgery.ufl.edu; Wang, Jin-Hee, E-mail: jin-hee.wang@surgery.ufl.edu; Biel, Thomas G., E-mail: Thomas.Biel@surgery.ufl.edu; Kim, Do-Sung, E-mail: do-sung.kim@surgery.med.ufl.edu; Flores-Toro, Joseph A., E-mail: Joseph.Flores-Toro@surgery.ufl.edu; Vijayvargiya, Richa, E-mail: rvijayvargiya@ufl.edu; Zendejas, Ivan, E-mail: ivan.zendejas@surgery.ufl.edu; Behrns, Kevin E., E-mail: Kevin.Behrns@surgery.ufl.edu

    2013-12-15

    Onset of the mitochondrial permeability transition (MPT) plays a causative role in ischemia/reperfusion (I/R) injury. Current therapeutic strategies for reducing reperfusion injury remain disappointing. Autophagy is a lysosome-mediated, catabolic process that timely eliminates abnormal or damaged cellular constituents and organelles such as dysfunctional mitochondria. I/R induces calcium overloading and calpain activation, leading to degradation of key autophagy-related proteins (Atg). Carbamazepine (CBZ), an FDA-approved anticonvulsant drug, has recently been reported to increase autophagy. We investigated the effects of CBZ on hepatic I/R injury. Hepatocytes and livers from male C57BL/6 mice were subjected to simulated in vitro, as well as in vivo I/R, respectively. Cell death, intracellular calcium, calpain activity, changes in autophagy-related proteins (Atg), autophagic flux, MPT and mitochondrial membrane potential after I/R were analyzed in the presence and absence of 20 μM CBZ. CBZ significantly increased hepatocyte viability after reperfusion. Confocal microscopy revealed that CBZ prevented calcium overloading, the onset of the MPT and mitochondrial depolarization. Immunoblotting and fluorometric analysis showed that CBZ blocked calpain activation, depletion of Atg7 and Beclin-1 and loss of autophagic flux after reperfusion. Intravital multiphoton imaging of anesthetized mice demonstrated that CBZ substantially reversed autophagic defects and mitochondrial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium overloading and calpain activation, which, in turn, suppresses Atg7 and Beclin-1 depletion, defective autophagy, onset of the MPT and cell death after I/R. - Highlights: • A mechanism of carbamazepine (CBZ)-induced cytoprotection in livers is proposed. • Impaired autophagy is a key event contributing to lethal reperfusion injury. • The importance of autophagy is extended and confirmed in an in vivo model. • CBZ is a potential

  17. Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy.

    Science.gov (United States)

    Zabirnyk, Olga; Liu, Wei; Khalil, Shadi; Sharma, Anit; Phang, James M

    2010-03-01

    Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.

  18. Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication.

    Science.gov (United States)

    Meng, Chunchun; Zhou, Zhizhi; Jiang, Ke; Yu, Shengqing; Jia, Lijun; Wu, Yantao; Liu, Yanqing; Meng, Songshu; Ding, Chan

    2012-06-01

    Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

  19. The Role of Reactive Oxygen Species and Autophagy in Periodontitis and Their Potential Linkage

    Directory of Open Access Journals (Sweden)

    Chengcheng Liu

    2017-06-01

    Full Text Available Periodontitis is a chronic inflammatory disease that causes damage to periodontal tissues, which include the gingiva, periodontal ligament, and alveolar bone. The major cause of periodontal tissue destruction is an inappropriate host response to microorganisms and their products. Specifically, a homeostatic imbalance between reactive oxygen species (ROS and antioxidant defense systems has been implicated in the pathogenesis of periodontitis. Elevated levels of ROS acting as intracellular signal transducers result in autophagy, which plays a dual role in periodontitis by promoting cell death or blocking apoptosis in infected cells. Autophagy can also regulate ROS generation and scavenging. Investigations are ongoing to elucidate the crosstalk mechanisms between ROS and autophagy. Here, we review the physiological and pathological roles of ROS and autophagy in periodontal tissues. The redox-sensitive pathways related to autophagy, such as mTORC1, Beclin 1, and the Atg12-Atg5 complex, are explored in depth to provide a comprehensive overview of the crosstalk between ROS and autophagy. Based on the current evidence, we suggest that a potential linkage between ROS and autophagy is involved in the pathogenesis of periodontitis.

  20. Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Dang, Shipeng; Xu, Huanbai; Xu, Congfeng; Cai, Wei; Li, Qian; Cheng, Yiji; Jin, Min; Wang, Ru-Xing; Peng, Yongde; Zhang, Yi; Wu, Changping; He, Xiaozhou; Wan, Bing; Zhang, Yanyun

    2014-07-01

    Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4(+) T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy.

  1. Chloroquine enhances the efficacy of cisplatin by suppressing autophagy in human adrenocortical carcinoma treatment

    Directory of Open Access Journals (Sweden)

    Qin L

    2016-03-01

    Full Text Available Liang Qin,1,* Tianyuan Xu,1,* Leilei Xia,1 Xianjin Wang,1 Xiang Zhang,1 Xiaohua Zhang,1 Zhaowei Zhu,1 Shan Zhong,1 Chuandong Wang,2 Zhoujun Shen1 1Department of Urology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 2Key Laboratory of Stem Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: It has been demonstrated that chloroquine (CQ enhances the efficacy of chemotherapy. However, little is known about whether CQ could enhance the efficacy of cisplatin (DDP in the treatment of adrenocortical carcinoma (ACC. In this study, we explore the efficacy and mechanism by which CQ affects DDP sensitivity in human ACC in vitro and in vivo.Methods: The autophagic gene Beclin-1 expression was detected by immunohistochemistry, and the protein levels were analyzed using immunoblotting assays of ACC tissues and normal adrenal cortex tissues. The ACC SW13 cells were treated with DDP and/or CQ. The cell viability assay was performed using the MTT method. Qualitative autophagy detection was performed by monodansylcadaverine staining of autophagic vacuoles. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was used to count cell apoptosis by flow cytometry. The autophagy-related protein (Beclin-1, LC3, and p62 and apoptosis relative protein (Bax and Bcl-2 levels were evaluated with Western blot analysis. Furthermore, a murine model of nude BALB/c mice bearing SW13 cell xenografts was established to evaluate the efficacy of concomitant therapy.Results: The expression of the autophagic gene Beclin-1 was significantly downregulated in ACC tissues compared to normal adrenal cortex tissues. The Beclin-1 protein level in ACC tissues was lower than that in normal adrenal cortex tissues (P<0.05. In vitro concomitant therapy (DDP and CQ was more

  2. Cardiomyocyte apoptosis vs autophagy with prolonged doxorubicin treatment: comparison with osteosarcoma cells.

    Science.gov (United States)

    Tacar, Oktay; Indumathy, Sivanjah; Tan, Mei Lin; Baindur-Hudson, Swati; Friedhuber, Anna M; Dass, Crispin R

    2015-02-01

    Doxorubicin (Dox) is a frontline chemotherapeutic against osteosarcoma (OS) that is plagued by side effects, particularly in the heart. The specific objective of this article is to investigate whether low-dose Dox treatment had pro-autophagic effects in cardiomyocytes as well as osteosarcoma cells. This study characterises apoptotic (Bax) and autophagic (Beclin-1) biomarker levels in human OS and cardiomyocyte cell lines as well as in various tissues when mice are exposed to low (1 mg/kg, thrice weekly) and high (3 mg/kg thrice weekly) dose Dox for a month. There was a decrease in Bax and increase in Beclin-1 in cardiac tissue in the high-dose group. Dox decreased Beclin-1 in the skin and liver, with no clear indication in the stomach, small intestine and testis. At low Dox doses of 10 and 100 nm in cardiomyocytes and OS cells, there is a pro-apoptotic effect, with a quicker response in the 100-nm condition, and a slower but steady increase of a pro-apoptotic response at the lower 10-nm dose. However, electron microscopy images revealed changes to human OS cells that resembled autophagy. Human prostate, breast and colorectal cells treated with 10-nm Dox showed ∼ 40% reduction in cell viability after 24 h. In culture, cells of both cardiomyocytes and OS revealed a predominant pro-apoptotic response at the expense of autophagy, although both seemed to be occurring in vivo. © 2014 Royal Pharmaceutical Society.

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

    International Nuclear Information System (INIS)

    Wu, Chun-Yan; Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng; Guo, Xiao-Zhong; Wang, Hua

    2011-01-01

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

  4. Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia

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    Ling Du

    2017-06-01

    Full Text Available Background: Autophagy plays an important role in cardiovascular disease. Controversy still exists regarding the effect of autophagy on ischemic/hypoxic myocardium. Cardiac shock wave therapy (CSWT is an effective alternative treatment for refractory ischemic heart disease. Whether CSWT can regulate cardiomyocyte autophagy under hypoxic conditions is not clear. We established a myocardial hypoxia model using the H9c2 cell line and performed shock waves (SWs treatment to evaluate the effect of SW on autophagy. Methods: The H9c2 cells were incubated under hypoxic conditions, and SW treatment was then performed at energies of 0.02, 0.05, or 0.10 mJ/mm2. The cell viability and intracellular ATP level were examined. Western blot analysis was used to assess the expression of LC3B, AMPK, mTOR, Beclin-1, Sirt1, and HIF-1α. Autophagic vacuoles were visualized by monodansylcadaverine staining. Results: After the 24-hour hypoxic period, cardiomyocyte viability and ATP levels were decreased and autophagy was significantly increased in H9c2 cells. SW treatment with an energy of 0.05 mJ/mm2 significantly increased the cellular viability, ATP level, LC3B-II/I, and number of autophagic vacuoles. In addition, phosphorylated AMPK and Sirt1 were increased and phosphorylated mTOR and HIF-1α were decreased after SW treatment. Conclusion: SW treatment can potentially promote cardiomyocyte autophagy during hypoxia and protect cardiomyocyte function by regulating the AMPK/mTOR pathway.

  5. 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.

  6. Vorinostat-induced autophagy switches from a death-promoting to a cytoprotective signal to drive acquired resistance.

    Science.gov (United States)

    Dupéré-Richer, D; Kinal, M; Ménasché, V; Nielsen, T H; Del Rincon, S; Pettersson, F; Miller, W H

    2013-02-07

    Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, de novo or acquired resistance to HDACi therapy is inevitable, and their molecular mechanisms are still unclear. To gain insight into HDACi resistance, we developed vorinostat-resistant clones from the hematological cell lines U937 and SUDHL6. Although cross-resistant to some but not all HDACi, the resistant cell lines exhibit dramatically increased sensitivity toward chloroquine, an inhibitor of autophagy. Consistent with this, resistant cells growing in vorinostat show increased autophagy. Inhibition of autophagy in vorinostat-resistant U937 cells by knockdown of Beclin-1 or Lamp-2 (lysosome-associated membrane protein 2) restores sensitivity to vorinostat. Interestingly, autophagy is also activated in parental U937 cells by de novo treatment with vorinostat. However, in contrast to the resistant cells, inhibition of autophagy decreases sensitivity to vorinostat. These results indicate that autophagy can switch from a proapoptotic signal to a prosurvival function driving acquired resistance. Moreover, inducers of autophagy (such as mammalian target of rapamycin inhibitors) synergize with vorinostat to induce cell death in parental cells, whereas the resistant cells remain insensitive. These data highlight the complexity of the design of combination strategies using modulators of autophagy and HDACi for the treatment of hematological malignancies.

  7. Autophagy in photodynamic therapy

    African Journals Online (AJOL)

    Autophagy is a conserved intracellular degradation process in which cellular organelles, proteins and invading microbes are degraded by lysosomes. There are three types of autophagy: macroautophagy, mitoautophagy and chaperone- mediated autophagy. This review is focused on macroautophagy which is referred to ...

  8. Beclin 1 overexpression inhibits chondrocyte apoptosis and downregulates extracellular matrix metabolism in osteoarthritis.

    Science.gov (United States)

    Song, Bin; Song, Hong; Wang, Weiguo; Wang, Hongru; Peng, Hanyuan; Cui, Jing; Wang, Rong; Huang, Hua; Wang, Wei; Wang, Lili

    2017-10-01

    In the present study, the expression of Beclin 1 in osteoarthritis (OA) cartilage tissue was investigated, and also its role in proliferation, apoptosis and expression of matrix metalloproteinases (MMPs) in chondrocytes obtained from patients with OA. Beclin 1 expression in cartilage tissue from OA patients, and in the age- and sex-matched controls, was detected by immunohistochemistry, semi-quantitative polymerase chain reaction and western blotting. Chondrocytes were divided into control and Beclin 1-overexpressed groups. After transfection for 48, 72 and 96 h, cell viability, apoptosis, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway and MMPs were examined. The mRNA and protein expression levels of Beclin 1 were significantly decreased in cartilage tissue from OA patients compared with the sex- and age-matched controls (Poverexpression significantly increased cell viability (Poverexpression additionally decreased the degree of apoptosis, as demonstrated by Hoechst staining and flow cytometric analysis. B-cell lymphoma-2 (Bcl-2) was upregulated, and Bcl-2 associated X was downregulated, following Beclin 1 overexpression (Poverexpression (Poverexpression (Poverexpression increased cell viability, inhibited apoptosis and MMPs, likely via the PI3K/Akt/mTOR signaling pathway.

  9. Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

    Science.gov (United States)

    Won, Shen-Jeu; Yen, Cheng-Hsin; Lin, Ting-Yu; Jiang-Shieh, Ya-Fen; Lin, Chun-Nan; Chen, Jyun-Ti; Su, Chun-Li

    2018-01-01

    The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer. © 2017 Wiley Periodicals, Inc.

  10. Starvation Promotes Autophagy-Associated Maturation of the Ovary in the Giant Freshwater Prawn, Macrobrachium rosenbergii

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    Wilairat Kankuan

    2017-05-01

    Full Text Available Limitation of food availability (starvation is known to influence the reproductive ability of animals. Autophagy is a lysosomal driven degradation process that protects the cell under metabolic stress conditions, such as during nutrient shortage. Whether, and how starvation-induced autophagy impacts on the maturation and function of reproductive organs in animals are still open questions. In this study, we have investigated the effects of starvation on histological and cellular changes that may be associated with autophagy in the ovary of the giant freshwater prawn, Macrobachium rosenbergii. To this end, the female prawns were daily fed (controls or unfed (starvation condition for up to 12 days, and the ovary tissue was analyzed at different time-points. Starvation triggered ovarian maturation, and concomitantly increased the expression of autophagy markers in vitellogenic oocytes. The immunoreactivities for autophagy markers, including Beclin1, LC3-II, and Lamp1, were enhanced in the late oocytes within the mature ovaries, especially at the vitellogenic stages. These markers co-localized with vitellin in the yolk granules within the oocytes, suggesting that autophagy induced by starvation could drive vitellin utilization, thus promoting ovarian maturation.

  11. Pachymic acid promotes induction of autophagy related to IGF-1 signaling pathway in WI-38 cells.

    Science.gov (United States)

    Lee, Su-Gyeong; Kim, Moon-Moo

    2017-12-01

    The insulin-like growth factor 1 (IGF-1) signaling pathway has spotlighted as a mechanism to elucidate aging associated with autophagy in recent years. Therefore, we have tried to screen an effective compound capable of inducing autophagy to delay aging process. The aim of this study is to investigate whether pachymic acid, a main compound in Poria cocos, induces autophagy in the aged cells. The aging of young cells was induced by treatment with IGF-1 at 50 ng/ml three times every two days. The effect of pachymic acid on cell viability was evaluated in human lung fibroblasts, WI-38 cells, using MTT assay. The induction of autophagy was detected using autophagy detection kit. The expression of proteins related to autophagy and IGF-1 signaling pathway was examined by western blot analysis and immunofluorescence assay. In this study, pachymic acid showed cytotoxic effect in a dose dependent manner and remarkably induced autophagy at the same time. Moreover, pachymic acid increased the expression of proteins related to autophagy such as LC3-II and Beclin1 and decreased the levels of mTor phosphorylation and p70S6K in the aged cells. In particular, pachymic acid increased the expression of p-PI3K, p-FoxO and Catalase. In addition, pachymic acid remarkably increased the expression of IGFBP-3. Above results suggest that pachymic acid could induce autophagy related to IGF-1 signaling pathway in the aged cells. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. Shuangshen Ningxin Capsule, a Traditional Chinese Medicinal Preparation, Alleviates Myocardial Ischemia through Autophagy Regulation

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    Jun Wang

    2015-01-01

    Full Text Available Shuangshen Ningxin capsule (SSNX, a modern Chinese formula, has been used to treat cardiovascular diseases in Eastern Asia. Our study focuses on the autophagy regulation of SSNX against coronary artery injuries. Myocardial infarction model was established in Chinese miniswines (CMS by coronary artery balloon injury. SSNX was administered to the CMS for 8 weeks with 4 mg/kg or 16 mg/kg. Myocardial cells were incubated with 20% SSNX medicated serum for 2 hours. Assays were performed to detect the effects of SSNX on (i coronary artery diameter by angiography, (ii hemodynamics by noninvasive hemodynamic monitoring system, (iii plaque burden and plaque volume by intravenous ultrasound (iv coronary artery histology by H&E staining, (v autophagosome by transmission electron microscopy, (vi lactate dehydrogenase (LDH leakage, and (vii Beclin-1 and LC3-I/II expressions by Western blot. The results showed that CMS treated with SSNX exhibited the correction for the disturbed cardiac hemodynamics, increase of coronary artery diameter, reduction of high plaque burden and plaque volume, and decrease of LDH. The inhibitory effect of SSNX on CMS autophagy was demonstrated by the reduction of autophagosome and the downregulation of beclin-1 and LC3-I/II. SSNX may protect coronary artery and increase the stability of plaque through the suppression of myocardial cellular autophagy, which suggests the potentially therapeutic effect of SSNX on ischemic cardiovascular disease.

  13. Autophagy influences the low-dose hyper-radiosensitivity of human lung adenocarcinoma cells by regulating MLH1.

    Science.gov (United States)

    Wang, Qiong; Xiao, Zhuya; Lin, Zhenyu; Zhou, Jie; Chen, Weihong; Jie, Wuyun; Cao, Xing; Yin, Zhongyuan; Cheng, Jing

    2017-06-01

    To investigate the impact of autophagy on the low-dose hyper-radiosensitivity (HRS) of human lung adenocarcinoma cells via MLH1 regulation. Immunofluorescent staining, Western blotting, and electron microscopy were utilized to detect autophagy in A549 and H460 cells. shRNA was used to silence MLH1 expression. The levels of MLH1, mTOR, p-mTOR, BNIP3, and Beclin-1 were measured by real-time polymerase chain reaction (PCR) and Western blotting. A549 cells, which have low levels of MLH1 expression, displayed HRS/induced radioresistance (IRR). Conversely, the radiosensitivity of H460 cells, which express high levels of MLH1, conformed to the linear-quadratic (LQ) model. After down-regulating MLH1 expression, A549 cells showed increased HRS and inhibition of autophagy, whereas H460 cells exhibited HRS/IRR. The levels of mTOR, p-mTOR, and BNIP3 were reduced in cells harboring MLH1 shRNA, and the changes in the mTOR/p-mTOR ratio mirrored those in MLH1 expression. Low MLH1-expressing A549 cells may exhibit HRS. Both the mTOR/p-mTOR and BNIP3/Beclin-1 signaling pathways were found to be related to HRS, but only mTOR/p-mTOR is involved in the regulation of HRS via MLH1 and autophagy.

  14. [Interleukin-37 induces apoptosis and autophagy of SMMC-7721 cells by inhibiting phosphorylation of mTOR].

    Science.gov (United States)

    Li, Tingting; Zhu, Di; Mou, Tong; Guo, Zhen; Pu, Junliang; Wu, Zhongjun

    2017-04-01

    Objective To investigate the underlying mechanism by which interleukin-37 (IL-37) induces the apoptosis and autophagy in SMMC-7721 cells. Methods SMMC-7721 cells were incubated in vitro and divided into two groups, IL-37 treated group and control group. The cells were treated with (50, 100, 200) ng/mL of recombinant human interleukin-37 (rhIL-37). CCK-8 assay was used to detect the cell proliferation of SMMC-7721 cells. Cell apoptosis was measured by flow cytometry. Western blot analysis was performed to examine the expressions of apoptosis-related proteins, Bax, Bcl-2, and autophagy related proteins, microtubule-associated proteins 1 light chain 3 (LC3), beclin 1 and mammalian target of rapamycin (mTOR). Transmission electron microscopy (TEM) was used to observe the ultrastructures of autophagosomes. Results The rhIL-37 inhibited the proliferation of hepatocellular carcinoma SMMC-7721 cells. It induced the apoptosis and autophagy in SMMC-7721 cells. In the IL-37 treated group, the levels of Bax, LC3 and beclin 1 increased but Bcl-2 decreased. The phosphorylation of mTOR was inhibited in the IL-37 treated group. Autophagosome was obvious in the IL-37 treated group. Conclusion IL-37 induces the apoptosis and autophagy in SMMC-7721 cells, which may be related to the phosphorylation of mTOR.

  15. 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.

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

    Science.gov (United States)

    Chandra, Dhyan

    2012-01-01

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

  17. Sorafenib-induced defective autophagy promotes cell death by necroptosis.

    Science.gov (United States)

    Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Björklund, Ann-Charlotte; Zhivotovsky, Boris; Grandér, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

    2015-11-10

    Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5-/- cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.

  18. Autophagy promotes degradation of polyethyleneimine–alginate nanoparticles in endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Wang GD

    2017-09-01

    Full Text Available Guo-dong Wang, Yu-zhen Tan, Hai-jie Wang, Pei Zhou Department of Anatomy, Histology and Embryology, Shanghai Medical School of Fudan University, Shanghai, China Abstract: Polyethyleneimine (PEI–alginate (Alg nanoparticle (NP is a safe and effective vector for delivery of siRNA or DNA. Recent studies suggest that autophagy is related to cytotoxicity of PEI NPs. However, contribution of autophagy to degradation of PEI–Alg NPs remains unknown. CD34+VEGFR-3+ endothelial progenitor cells isolated from rat bone marrow were treated with 25 kDa branched PEI modified by Alg. After treatment with the NPs, morphological changes and distribution of the NPs in the cells were examined with scanning and transmission electron microscopies. Cytotoxicity of the NPs was analyzed by reactive oxygen species (ROS production, lactate dehydrogenase leakage and induction of apoptosis. The level of autophagy was assessed with expression of Beclin-1 and LC3 and formation of autophagic structures and amphisomes. Colocalization of LC3-positive puncta and the NPs was determined by LC3–GFP tracing. Cytotoxicity of PEI NPs was reduced greatly after modification with Alg. PEI–Alg NPs were distributed in mitochondria, rough endoplasmic reticula and nuclei as well as cytoplasm. After phagocytosis of the NPs, expression of Beclin-1 mRNA and LC3 protein was upregulated, and the number of LC3-positive puncta, autophagic structures and amphisomes increased significantly. The number of lysosomes also increased obviously. There were LC3-positive puncta in nuclei, and some puncta were colocalized with the NPs. These results demonstrate that the activated autophagy promotes degradation of PEI–Alg NPs via multiple pathways. Keywords: polyethyleneimine, alginate, nanoparticles, endothelial progenitor cells, autophagy

  19. The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes.

    Science.gov (United States)

    Yin, Yiran; Tang, Lian; Shi, Lei

    2017-03-01

    The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (Posteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.

  20. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains▿ †

    OpenAIRE

    Bellot, Grégory; Garcia-Medina, Raquel; Gounon, Pierre; Chiche, Johanna; Roux, Danièle; Pouysségur, Jacques; Mazure, Nathalie M.

    2009-01-01

    While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy,...

  1. Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells.

    Science.gov (United States)

    Lin, Ji-Fan; Lin, Yi-Chia; Tsai, Te-Fu; Chen, Hung-En; Chou, Kuang-Yu; Hwang, Thomas I-Sheng

    2017-01-01

    Cisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines. Human BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation. Cisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis. Collectively, the study results

  2. The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells

    International Nuclear Information System (INIS)

    Li, Jun; Qin, Zhenghong; Liang, Zhongqin

    2009-01-01

    Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (ΔΨm) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol. Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells

  3. GLP-1 analogue improves hepatic lipid accumulation by inducing autophagy via AMPK/mTOR pathway

    Energy Technology Data Exchange (ETDEWEB)

    He, Qin; Sha, Sha; Sun, Lei; Zhang, Jing; Dong, Ming, E-mail: dr_dongming@126.com

    2016-08-05

    The incidence of nonalcoholic fatty liver disease (NAFLD) keeps rising year by year, and NAFLD is rapidly becoming the most common liver disease worldwide. Clinical studies have found that glucagon-like peptide-1 (GLP-1) analogue, liraglutide (LRG), cannot only reduce glucose levels, but also improve hepatic lipase, especially in patients also with type 2 diabetes mellitus (T2DM). In addition, enhancing autophagy decreases lipid accumulation in hepatocytes. The aim of the present study is to explore the effect of LRG on hepatocyte steatosis and the possible role of autophagy. We set up an obesity mouse model with a high-fat diet (HFD) and induced hepatocyte steatosis with free fatty acids (FFA) in human L-O2 cells. LRG and two inhibitors of autophagy, Chloroquine (CQ) and bafilomycin A1 (Baf), were added into each group, respectively. The lipid profiles and morphological modifications of each group were tested. Immunohistochemistry, immunofluorescence staining and transmission electron microscopy (TEM) were used to measure autophagy in this study. The autophagy protein expression of SQSTM1 (P62), and LC3B, along with the signaling pathway proteins of mTOR, phosphorylated mTOR (p-mTOR), AMPK, phosphorylated AMPK (p-AMPK) and Beclin1, were evaluated by western blot. Our results showed that LRG improved hepatocyte steatosis by inducing autophagy, and the AMPK/mTOR pathway is involved. These findings suggest an important mechanism for the positive effects of LRG on hepatic steatosis, and provide new evidence for clinical use of LRG in NAFLD. -- Highlights: •Liraglutide reduces lipid accumulation in hepatic steatosis both in vivo and in vitro. •Autophagy was involved in relieving effects of liraglutide on hepatic steatosis. •AMPK/mTOR pathway was involved in liraglutide-induced autophagy.

  4. GLP-1 analogue improves hepatic lipid accumulation by inducing autophagy via AMPK/mTOR pathway

    International Nuclear Information System (INIS)

    He, Qin; Sha, Sha; Sun, Lei; Zhang, Jing; Dong, Ming

    2016-01-01

    The incidence of nonalcoholic fatty liver disease (NAFLD) keeps rising year by year, and NAFLD is rapidly becoming the most common liver disease worldwide. Clinical studies have found that glucagon-like peptide-1 (GLP-1) analogue, liraglutide (LRG), cannot only reduce glucose levels, but also improve hepatic lipase, especially in patients also with type 2 diabetes mellitus (T2DM). In addition, enhancing autophagy decreases lipid accumulation in hepatocytes. The aim of the present study is to explore the effect of LRG on hepatocyte steatosis and the possible role of autophagy. We set up an obesity mouse model with a high-fat diet (HFD) and induced hepatocyte steatosis with free fatty acids (FFA) in human L-O2 cells. LRG and two inhibitors of autophagy, Chloroquine (CQ) and bafilomycin A1 (Baf), were added into each group, respectively. The lipid profiles and morphological modifications of each group were tested. Immunohistochemistry, immunofluorescence staining and transmission electron microscopy (TEM) were used to measure autophagy in this study. The autophagy protein expression of SQSTM1 (P62), and LC3B, along with the signaling pathway proteins of mTOR, phosphorylated mTOR (p-mTOR), AMPK, phosphorylated AMPK (p-AMPK) and Beclin1, were evaluated by western blot. Our results showed that LRG improved hepatocyte steatosis by inducing autophagy, and the AMPK/mTOR pathway is involved. These findings suggest an important mechanism for the positive effects of LRG on hepatic steatosis, and provide new evidence for clinical use of LRG in NAFLD. -- Highlights: •Liraglutide reduces lipid accumulation in hepatic steatosis both in vivo and in vitro. •Autophagy was involved in relieving effects of liraglutide on hepatic steatosis. •AMPK/mTOR pathway was involved in liraglutide-induced autophagy.

  5. Increased activity of osteocyte autophagy in ovariectomized rats and its correlation with oxidative stress status and bone loss

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yuehua, E-mail: yuesjtu@126.com; Zheng, Xinfeng, E-mail: zxf272@126.com; Li, Bo, E-mail: libo@126.com; Jiang, Shengdan, E-mail: jiangsd@126.com; Jiang, Leisheng, E-mail: leisheng_jiang@126.com

    2014-08-15

    Highlights: • Examine autophagy level in the proximal tibia of ovariectomized rats. • Investigate whether autophagy level is associated with bone loss. • Investigate whether autophagy level is associated with oxidative stress status. - Abstract: Objectives: The objectives of the present study were to investigate ovariectomy on autophagy level in the bone and to examine whether autophagy level is associated with bone loss and oxidative stress status. Methods: 36 female Sprague–Dawley rats were randomly divided into sham-operated (Sham), and ovariectomized (OVX) rats treated either with vehicle or 17-β-estradiol. At the end of the six-week treatment, bone mineral density (BMD) and bone micro-architecture in proximal tibias were assessed by micro-CT. Serum 17β-estradiol (E2) level were measured. Total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT) activity in proximal tibia was also determined. The osteocyte autophagy in proximal tibias was detected respectively by Transmission Electron Microscopy (TEM), immunofluorescent histochemistry (IH), realtime-PCR and Western blot. In addition, the spearman correlation between bone mass, oxidative stress status, serum E2 and autophagy were analyzed. Results: Ovariectomy increased Atg5, LC3, and Beclin1 mRNA and proteins expressions while decreased p62 expression. Ovariectomy also declined the activities of T-AOC, CAT, and SOD. Treatment with E2 prevented the reduction in bone mass as well as restored the autophagy level. Furthermore, LC3-II expression was inversely correlated with T-AOC, CAT, and SOD activities. A significant inverse correlation between LC3-II expression and BV/TV, Tb.N, BMD in proximal tibias was found. Conclusions: Ovariectomy induced oxidative stress, autophagy and bone loss. Autophagy of osteocyte was inversely correlated with oxidative stress status and bone loss.

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

    International Nuclear Information System (INIS)

    Liu, Jing; Zhang, Ye; Qu, Jinglei; Xu, Ling; Hou, Kezuo; Zhang, Jingdong; Qu, Xiujuan; Liu, Yunpeng

    2011-01-01

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

  7. BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells.

    Science.gov (United States)

    Liu, Bao-Qin; Du, Zhen-Xian; Zong, Zhi-Hong; Li, Chao; Li, Ning; Zhang, Qiang; Kong, De-Hui; Wang, Hua-Qin

    2013-06-01

    Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.

  8. Shear Stress Induces Phenotypic Modulation of Vascular Smooth Muscle Cells via AMPK/mTOR/ULK1-Mediated Autophagy.

    Science.gov (United States)

    Sun, Liqian; Zhao, Manman; Liu, Aihua; Lv, Ming; Zhang, Jingbo; Li, Youxiang; Yang, Xinjian; Wu, Zhongxue

    2018-03-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) is involved in the pathophysiological processes of the intracranial aneurysms (IAs). Although shear stress has been implicated in the proliferation, migration, and phenotypic conversion of VSMCs, the molecular mechanisms underlying these events are currently unknown. In this study, we investigated whether shear stress(SS)-induced VSMC phenotypic modulation was mediated by autophagy involved in adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway. The results show that shear stress could inhibit the expression of key VSMC contractile genes and induce pro-inflammatory/matrix-remodeling genes levels, contributing to VSMCs phenotypic switching from a contractile to a synthetic phenotype. More importantly, Shear stress also markedly increased the levels of the autophagy marker microtubule-associated protein light chain 3-II (LC3II), Beclin-1, and p62 degradation. The autophagy inhibitor 3-methyladenine (3-MA) significantly blocked shear-induced phenotypic modulation of VSMCs. To further explore the molecular mechanism involved in shear-induced autophagy, we found that shear stress could activate AMPK/mTOR/ULK1 signaling pathway in VSMCs. Compound C, a pharmacological inhibitor of AMPK, significantly reduced the levels of p-AMPK and p-ULK, enhanced p-mTOR level, and finally decreased LC3II and Beclin-1 level, which suggested that activated AMPK/mTOR/ULK1 signaling was related to shear-mediated autophagy. These results indicate that shear stress promotes VSMC phenotypic modulation through the induction of autophagy involved in activating the AMPK/mTOR/ULK1 pathway.

  9. Immunologic manifestations of autophagy

    DEFF Research Database (Denmark)

    Deretic, Vojo; Kimura, Tomonori; Timmins, Graham

    2015-01-01

    The broad immunologic roles of autophagy span innate and adaptive immunity and are often manifested in inflammatory diseases. The immune effects of autophagy partially overlap with its roles in metabolism and cytoplasmic quality control but typically expand further afield to encompass unique...... immunologic adaptations. One of the best-appreciated manifestations of autophagy is protection against microbial invasion, but this is by no means limited to direct elimination of intracellular pathogens and includes a stratified array of nearly all principal immunologic processes. This Review summarizes...... the broad immunologic roles of autophagy. Furthermore, it uses the autophagic control of Mycobacterium tuberculosis as a paradigm to illustrate the breadth and complexity of the immune effects of autophagy....

  10. Contribution of Hippocampal 5-HT3 Receptors in Hippocampal Autophagy and Extinction of Conditioned Fear Responses after a Single Prolonged Stress Exposure in Rats.

    Science.gov (United States)

    Wu, Zhong-Min; Yang, Li-Hua; Cui, Rong; Ni, Gui-Lian; Wu, Feng-Tian; Liang, Yong

    2017-05-01

    One of the hypotheses about the pathogenesis of posttraumatic stress disorder (PTSD) is the dysfunction of serotonin (5-HT) neurotransmission. While certain 5-HT receptor subtypes are likely critical for the symptoms of PTSD, few studies have examined the role of 5-HT 3 receptor in the development of PTSD, even though 5-HT 3 receptor is critical for contextual fear extinction and anxiety-like behavior. Therefore, we hypothesized that stimulation of 5-HT 3 receptor in the dorsal hippocampus (DH) could prevent hippocampal autophagy and the development of PTSD-like behavior in animals. To this end, we infused SR57227, selective 5-HT 3 agonist, into the DH after a single prolonged stress (SPS) treatment in rats. Three weeks later, we evaluated the effects of this pharmacological treatment on anxiety-related behaviors and extinction of contextual fear memory. We also accessed hippocampal autophagy and the expression of 5-HT 3A subunit, Beclin-1, LC3-I, and LC3-II in the DH. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT 3A expression, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. Furthermore, intraDH infusions of SR57227 dose-dependently promoted the extinction of contextual fear memory, prevented hippocampal autophagy, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. These results indicated that 5-HT 3 receptor in the hippocampus may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the pathophysiology of PTSD.

  11. Autophagy induction by Bcr-Abl-expressing cells facilitates their recovery from a targeted or nontargeted treatment.

    LENUS (Irish Health Repository)

    Crowley, Lisa C

    2012-01-31

    Although Imatinib has transformed the treatment of chronic myeloid leukemia (CML), it is not curative due to the persistence of resistant cells that can regenerate the disease. We have examined how Bcr-Abl-expressing cells respond to two mechanistically different therapeutic agents, etoposide and Imatinib. We also examined Bcr-Abl expression at low and high levels as elevated expression has been associated with treatment failure. Cells expressing low levels of Bcr-Abl undergo apoptosis in response to the DNA-targeting agent (etoposide), whereas high-Bcr-Abl-expressing cells primarily induce autophagy. Autophagic populations engage a delayed nonapoptotic death; however, sufficient cells evade this and repopulate following the withdrawal of the drug. Non-Bcr-Abl-expressing 32D or Ba\\/F3 cells induce both apoptosis and autophagy in response to etoposide and can recover. Imatinib treatment induces both apoptosis and autophagy in all Bcr-Abl-expressing cells and populations rapidly recover. Inhibition of autophagy with ATG7 and Beclin1 siRNA significantly reduced the recovery of Imatinib-treated K562 cells, indicating the importance of autophagy for the recovery of treated cells. Combination regimes incorporating agents that disrupt Imatinib-induced autophagy would remain primarily targeted and may improve response to the treatment in CML.

  12. Interplay between autophagy and apoptosis in lead(II)-induced cytotoxicity of primary rat proximal tubular cells.

    Science.gov (United States)

    Chu, Bing-Xin; Fan, Rui-Feng; Lin, Shu-Qian; Yang, Du-Bao; Wang, Zhen-Yong; Wang, Lin

    2018-05-01

    Autophagy and apoptosis are two different biological processes that determine cell fates. We previously reported that autophagy inhibition and apoptosis induction are involved in lead(II)-induced cytotoxicity in primary rat proximal tubular (rPT) cells, but the interplay between them remains to be elucidated. Firstly, data showed that lead(II)-induced elevation of LC3-II protein levels can be significantly modulated by 3-methyladenine or rapamycin; moreover, protein levels of Autophagy-related protein 5 (Atg5) and Beclin-1 were markedly up-regulated by lead(II) treatment, demonstrating that lead(II) could promote the autophagosomes formation in rPT cells. Next, we applied three pharmacological agents and genetic method targeting the early stage of autophagy to validate that enhancement of autophagosomes formation can inhibit lead(II)-induced apoptotic cell death in rPT cells. Simultaneously, lead(II) inhibited the autophagic degradation of rPT cells, while the addition of autophagic degradation inhibitor bafilomycin A1 aggravated lead(II)-induced apoptotic death in rPT cells. Collectively, this study provided us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells, and the interplay between autophagy and apoptosis highlights a new sight into the mechanism of lead(II)-induced nephrotoxicity. Copyright © 2018. Published by Elsevier Inc.

  13. Administration of Ketamine Causes Autophagy and Apoptosis in the Rat Fetal Hippocampus and in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Xinran Li

    2018-02-01

    Full Text Available Drug abuse during pregnancy is a serious problem. Like alcohol, anticonvulsants, sedatives, and anesthetics, such as ketamine, can pass through the placental barrier and affect the growing fetus. However, the mechanism by which ketamine causes damage to fetal rats is not well understood. Therefore, in this study, we anesthetized pregnant rats with ketamine and evaluated the Total Antioxidant Capacity (T-AOC, Reactive Oxygen Species (ROS, and Malondialdehyde (MDA. Moreover, we determined changes in the levels of Cleaved-Caspase-3 (C-Caspase-3, Beclin-1, B-cell lymphoma-2 (Bcl-2, Bcl-2 Associated X Protein (Bax, Autophagy-related gene 4 (Atg4, Atg5, p62 (SQSTM1, and marker of autophagy Light Chain 3 (LC3. In addition, we cultured PC12 cells in vitro to determine the relationship between ROS, autophagy, and apoptosis following ketamine treatment. The results showed that ketamine induced changes in autophagy- and apoptosis-related proteins, reduced T-AOC, and generated excessive levels of ROS and MDA. In vitro experiments showed similar results, indicating that apoptosis levels can be inhibited by 3-MA. We also found that autophagy and apoptosis can be inhibited by N-acetyl-L-cysteine (Nac. Thus, anesthesia with ketamine in pregnant rats may increase the rate of autophagy and apoptosis in the fetal hippocampus and the mechanism may be through inhibition of antioxidant activity and ROS accumulation.

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

    Science.gov (United States)

    Del Bello, Barbara; Toscano, Marzia; Moretti, Daniele; Maellaro, Emilia

    2013-01-01

    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.

  15. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells.

    Science.gov (United States)

    Tang, Zheng-Hai; Cao, Wen-Xiang; Wang, Zhao-Yu; Lu, Jia-Hong; Liu, Bo; Chen, Xiuping; Lu, Jin-Jian

    2017-08-01

    Chelerythrine (CHE), a natural benzo[c]phenanthridine alkaloid, shows anti-cancer effect through a number of mechanisms. Herein, the effect and mechanism of the CHE-induced autophagy, a type II programmed cell death, in non-small cell lung cancer (NSCLC) cells were studied for the first time. CHE induced cell viability decrease, colony formation inhibition, and apoptosis in a concentration-dependent manner in NSCLC A549 and NCI-H1299 cells. In addition, CHE triggered the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II). The CHE-induced expression of LC3-II was further increased in the combination treatment with chloroquine (CQ), an autophagy inhibitor, and large amounts of red-puncta were observed in the CHE-treated A549 cells with stable expression of mRFP-EGFP-LC3, indicating that CHE induces autophagy flux. Silence of beclin 1 reversed the CHE-induced expression of LC3-II. Inhibition of autophagy remarkably reversed the CHE-induced cell viability decrease and apoptosis in NCI-H1299 cells but not in A549 cells. Furthermore, CHE triggered reactive oxygen species (ROS) generation in both cell lines. A decreased level of ROS through pretreatment with N-acetyl-L-cysteine reversed the CHE-induced cell viability decrease, apoptosis, and autophagy. Taken together, CHE induced distinctive autophagy in A549 (accompanied autophagy) and NCI-H1299 (pro-death autophagy) cells and a decreased level of ROS reversed the effect of CHE in NSCLC cells in terms of cell viability, apoptosis, and autophagy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Sobhakumari, Arya; Schickling, Brandon M.; Love-Homan, Laurie; Raeburn, Ayanna; Fletcher, Elise V.M.; Case, Adam J.; Domann, Frederick E.; Miller, Francis J.

    2013-01-01

    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

  17. 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.

  18. Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy

    Directory of Open Access Journals (Sweden)

    Miao Shen

    2013-01-01

    Full Text Available Background. Hepatic ischemia-reperfusion (I/R injury is a pivotal clinical problem occurring in many clinical conditions such as transplantation, trauma, and hepatic failure after hemorrhagic shock. Apoptosis and autophagy have been shown to contribute to cell death in hepatic I/R injury. Ethyl pyruvate, a stable and simple lipophilic ester, has been shown to have anti-inflammatory properties. In this study, the purpose is to explore both the effect of ethyl pyruvate on hepatic I/R injury and regulation of intrinsic pathway of apoptosis and autophagy. Methods. Three doses of ethyl pyruvate (20 mg/kg, 40 mg/kg, and 80 mg/kg were administered 1 h before a model of segmental (70% hepatic warm ischemia was established in Balb/c mice. All serum and liver tissues were obtained at three different time points (4 h, 8 h, and 16 h. Results. Alanine aminotransferase (ALT, aspartate aminotransferase (AST, and pathological features were significantly ameliorated by ethyl pyruvate (80 mg/kg. The expression of Bcl-2, Bax, Beclin-1, and LC3, which play an important role in the regulation of intrinsic pathway of apoptosis and autophagy, was also obviously decreased by ethyl pyruvate (80 mg/kg. Furthermore, ethyl pyruvate inhibited the HMGB1/TLR4/ NF-κb axis and the release of cytokines (TNF-α and IL-6. Conclusion. Our results showed that ethyl pyruvate might attenuate to hepatic I/R injury by inhibiting intrinsic pathway of apoptosis and autophagy, mediated partly through downregulation of HMGB1/TLR4/ NF-κb axis and the competitive interaction with Beclin-1 of HMGB1.

  19. A role for autophagy in long-term spatial memory formation in male rodents.

    Science.gov (United States)

    Hylin, Michael J; Zhao, Jing; Tangavelou, Karthikeyan; Rozas, Natalia S; Hood, Kimberly N; MacGowan, Jacalyn S; Moore, Anthony N; Dash, Pramod K

    2018-03-01

    A hallmark of long-term memory formation is the requirement for protein synthesis. Administration of protein synthesis inhibitors impairs long-term memory formation without influencing short-term memory. Rapamycin is a specific inhibitor of target of rapamycin complex 1 (TORC1) that has been shown to block protein synthesis and impair long-term memory. In addition to regulating protein synthesis, TORC1 also phosphorylates Unc-51-like autophagy activating kinase-1 (Ulk-1) to suppress autophagy. As autophagy can be activated by rapamycin (and rapamycin inhibits long-term memory), our aim was to test the hypothesis that autophagy inhibitors would enhance long-term memory. To examine if learning alters autophagosome number, we used male reporter mice carrying the GFP-LC3 transgene. Using these mice, we observed that training in the Morris water maze task increases the number of autophagosomes, a finding contrary to our expectations. For learning and memory studies, male Long Evans rats were used due to their relatively larger size (compared to mice), making it easier to perform intrahippocampal infusions in awake, moving animals. When the autophagy inhibitors 3-methyladenine (3-MA) or Spautin-1 were administered bilaterally into the hippocampii prior to training in the Morris water maze task, the drugs did not alter learning. In contrast, when memory was tested 24 hours later by a probe trial, significant impairments were observed. In addition, intrahippocampal infusion of an autophagy activator peptide (TAT-Beclin-1) improved long-term memory. These results indicate that autophagy is not necessary for learning, but is required for long-term memory formation. © 2017 Wiley Periodicals, Inc.

  20. Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

    International Nuclear Information System (INIS)

    Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming

    2016-01-01

    The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

  1. Effect of limb demand ischemia on autophagy and morphology in mice.

    Science.gov (United States)

    Albadawi, Hassan; Oklu, Rahmi; Milner, John D; Uong, Thuy P; Yoo, Hyung-Jin; Austen, William G; Watkins, Michael T

    2015-10-01

    Obesity is a major risk factor for diabetes and peripheral arterial disease, which frequently leads to lower limb demand ischemia. Skeletal muscle autophagy and mitochondrial biogenesis are important processes for proper oxidative capacity and energy metabolism, which are compromised in diabetes. This study compares autophagy, mitochondrial biogenesis, energy metabolism, and morphology in the hind limbs of obese diabetic mice subjected to demand or sedentary ischemia. Unilateral hind limb demand ischemia was created in a group of diet-induced obese mice after femoral artery ligation and 4 wk of daily exercise. A parallel group of mice underwent femoral artery ligation but remained sedentary for 4 wk. Hind limb muscles were analyzed for markers of autophagy, mitochondrial biogenesis, adenosine triphosphate, and muscle tissue morphology. At the end of the 4-wk exercise period, demand ischemia increased the autophagy mediator Beclin-1, but it did not alter the autophagy indicator, LC3B-II/I ratio, or markers of mitochondrial biogenesis, optic atrophy/dynamin-related protein. In contrast, exercise significantly increased the level of mitochondrial protein-succinate dehydrogenase subunit-A and reduced adipocyte accumulation and the percentage of centrally nucleated myofibers in the demand ischemia limb. In addition, demand ischemia resulted in decreased uncoupling protein-3 levels without altering muscle adenosine triphosphate or pS473-Akt levels. Limb demand ischemia markedly decreased adipocyte accumulation and enhanced muscle regeneration in obese mice, but it did not appear to enhance autophagy, mitochondrial biogenesis, energy metabolism, or insulin sensitivity. Future studies aimed at evaluating novel therapies that enhance autophagy and mitochondrial biogenesis in diabetes with peripheral arterial disease are warranted. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming, E-mail: zengshenming@gmail.com

    2016-04-22

    The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

  3. Autophagy in protists

    Science.gov (United States)

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

    2011-01-01

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

  4. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

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    Jintao Zhang

    Full Text Available Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.Human colorectal cancer cell lines (HCT-116 and HT-29 were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining, and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II, beclin-1, and autophagocytosis-associated protein (Atg3. The autophagy inhibitors 3-methyladenine (3-MA and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin and genetic

  5. Andrographolide Induces Autophagic Cell Death and Inhibits Invasion and Metastasis of Human Osteosarcoma Cells in An Autophagy-Dependent Manner

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    Ying Liu

    2017-11-01

    Full Text Available Background/Aims: Osteosarcoma (OS is the most common primary malignant tumor of bone tissue. Although treatment effectiveness has improved, the OS survival rate has fluctuated in recent years. Andrographolide (AG has been reported to have antitumor activity against a variety of tumors. Our aim was to investigate the effects and potential mechanisms of AG in human osteosarcoma. Methods: Cell viability and morphological changes were assessed by MTT and live/dead assays. Apoptosis was detected using Annexin V-FITC/PI double staining, DAPI, and caspase-3 assays. Autophagy was detected with mRFP-GFP-LC3 adenovirus transfection and western blot. Cell migration and invasion were detected by wound healing assay and Transwell® experiments. Results: AG dose-dependently reduced the viability of osteosarcoma cells. No increase in apoptosis was detected in AG-treated human OS MG-63 and U-2OS cells, and the pan-caspase inhibitor z-VAD did not attenuate AG-induced cell death. However, AG induced autophagy by suppressing PI3K/Akt/mTOR and enhancing JNK signaling pathways. 3-MA and Beclin-1 siRNA could reverse the cytotoxic effects of AG. In addition, AG inhibited the invasion and metastasis of OS, and this effect could be reversed with Beclin-1 siRNA. Conclusion: AG inhibits viability and induces autophagic death in OS cells. AG-induced autophagy inhibits the invasion and metastasis of OS.

  6. [TLR2 modulates Staphylococcus aureus-induced inflammatory response and autophagy in macrophages through PI3K signaling pathway].

    Science.gov (United States)

    Li, Shuai; Fang, Lei; Wang, Jiong; Liu, Rongyu

    2017-09-01

    Objective To investigate the molecular mechanisms of Toll-like receptor 2 (TLR2) taking part in inflammatory response in Staphylococcus aureus (SA)-induced asthma. Methods We established the cell inflammatory response model through stimulating mouse RAW264.7 macrophages with SA. The TLR2, myeloid differentiation factor 88 (MyD88), phosphoinositide-3 kinase (PI3K), nuclear factor κBp65 (NF-κBp65), phospho-NF-κBp65, beclin-1 and microtubule-associated protein 1 light chain 3B (LC3B) were detected by Western blot analysis after treatment with TLR2 small interfering RNA (siRNA) and 3-methyladenine (3-MA), and the tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were determined by ELISA. In addition, the number of autolysosomes was observed by the laser scanning confocal microscope. Results SA-stimulated macrophages activated various signaling pathways including TLR2. TLR2 siRNA markedly repressed the expressions of PI3K, phospho-NF-κBp65, the autophagy protein beclin-1 and LC3B as well as the number of autolysosomes and the production of TNF- and IL-6. We also demonstrated that 3-MA had the same effect on autophagy and inflammation as TLR2 siRNA did. Conclusion TLR2 modulates SA-induced inflammatory response and autophagy in macrophages through PI3K signaling pathway.

  7. [Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells].

    Science.gov (United States)

    Wang, Jing; Wang, Jin; Wang, Juan-Juan; Zhang, Wei-Fang; Jiao, Xiang-Ying

    2017-08-25

    Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.

  8. Impacts of autophagy-inducing ingredient of areca nut on tumor cells.

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    Ching-Yu Yen

    Full Text Available Areca nut (AN is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE and its 30-100 kDa fraction (ANE 30-100K predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth, CE81T/VGH (esophagus, SCC25 (tongue, and SCC-15 (tongue. The results showed that chemical- and small hairpin RNA (shRNA-mediated inhibition of AMP-activated protein kinase (AMPK resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.

  9. Green Tea Polyphenols, Mimicking the Effects of Dietary Restriction, Ameliorate High-Fat Diet-Induced Kidney Injury via Regulating Autophagy Flux

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    Xiao Xie

    2017-05-01

    Full Text Available Epidemiological and experimental studies reveal that Western dietary patterns contribute to chronic kidney disease, whereas dietary restriction (DR or dietary polyphenols such as green tea polyphenols (GTPs can ameliorate the progression of kidney injury. This study aimed to investigate the renal protective effects of GTPs and explore the underlying mechanisms. Sixty Wistar rats were randomly divided into 6 groups: standard diet (STD, DR, high-fat diet (HFD, and three diets plus 200 mg/kg(bw/day GTPs, respectively. After 18 weeks, HFD group exhibited renal injuries by increased serum cystatin C levels and urinary N-acetyl-β-d-glucosaminidase activity, which can be ameliorated by GTPs. Meanwhile, autophagy impairment as denoted by autophagy-lysosome related proteins, including LC3-II, Beclin-1, p62, cathepsin B, cathepsin D and LAMP-1, was observed in HFD group, whereas DR or GTPs promoted renal autophagy activities and GTPs ameliorated HFD-induced autophagy impairment. In vitro, autophagy flux suppression was detected in palmitic acid (PA-treated human proximal tubular epithelial cells (HK-2, which was ameliorated by epigallocatechin-3-gallate (EGCG. Furthermore, GTPs (or EGCG elevated phosphorylation of AMP-activated protein kinase in the kidneys of HFD-treated rats and in PA-treated HK-2 cells. These findings revealed that GTPs mimic the effects of DR to induce autophagy and exert a renal protective effect by alleviating HFD-induced autophagy suppression.

  10. Dengue Virus and Autophagy

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    Nicholas S. Heaton

    2011-08-01

    Full Text Available Several independent groups have published that autophagy is required for optimal RNA replication of dengue virus (DENV. Initially, it was postulated that autophagosomes might play a structural role in replication complex formation. However, cryo-EM tomography of DENV replication complexes showed that DENV replicates on endoplasmic reticulum (ER cisternae invaginations and not on classical autophagosomes. Recently, it was reported that autophagy plays an indirect role in DENV replication by modulating cellular lipid metabolism. DENV-induced autophagosomes deplete cellular triglycerides that are stored in lipid droplets, leading to increased β-oxidation and energy production. This is the first example of a virus triggering autophagy to modulate cellular physiology. In this review, we summarize these data and discuss new questions and implications for autophagy during DENV replication.

  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. Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

    Science.gov (United States)

    Pan, W-R; Chen, P-W; Chen, Y-L S; Hsu, H-C; Lin, C-C; Chen, W-J

    2013-01-01

    Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 μM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Autophagy in Trypanosomatids

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    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. The ubiquitin-proteasome system and autophagy are defective in the taurine-deficient heart.

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    Jong, Chian Ju; Ito, Takashi; Schaffer, Stephen W

    2015-12-01

    Taurine depletion leads to impaired mitochondrial function, as characterized by reduced ATP production and elevated superoxide generation. These defects can fundamentally alter cardiomyocyte function and if left unchanged can result in cell death. To protect against these stresses, cardiomyocytes possess quality control processes, such as the ubiquitin-proteasome system (UPS) and autophagy, which can rejuvenate cells through the degradation of damaged proteins and organelles. Hence, the present study tested the hypothesis that reactive oxygen species generated by damaged mitochondria initiates UPS and autophagy in the taurine-deficient heart. Using transgenic mice lacking the taurine transporter (TauTKO) as a model of taurine deficiency, it was shown that the levels of ubiquitinated protein were elevated, an effect associated with a decrease in ATP-dependent 26S β5 proteasome activity. Treating the TauTKO mouse with the mitochondria-specific antioxidant, mitoTEMPO, largely abolished the increase in ubiquitinated protein content. The TauTKO heart was also associated with impaired autophagy, characterized by an increase in the initiator, Beclin-1, and autophagosome content, but a defect in the generation of active autophagolysosomes. Although mitoTEMPO treatment only restores the oxidative balance within the mitochondria, it appeared to completely disrupt the crosstalk between the damaged mitochondria and the quality control processes. Thus, mitochondrial oxidative stress is the main trigger initiating the quality control systems in the taurine-deficient heart. We conclude that the activation of the UPS and autophagy is another fundamental function of mitochondria.

  15. High Autophagy in the Naked Mole Rat may Play a Significant Role in Maintaining Good Health

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    Shanmin Zhao

    2014-02-01

    Full Text Available Background/Aims: The maximum lifespan of the naked mole rat is over 28.3 years, which exceeds that of any other rodent species, suggesting that age-related changes in its body composition and functionality are either attenuated or delayed in this extraordinarily long-lived species. However, the mechanisms underlying the aging process in this species are poorly understood. In this study, we investigated whether long-lived naked mole rats display more autophagic activity than short-lived mice. Methods: Hepatic stellate cells isolated from naked mole rats were treated with 50 nM rapamycin or 20 mM 3-methyladenine (3-MA for 12 or 24 h. Expression of the autophagy marker proteins LC3-II and beclin 1 was measured with western blotting and immunohistochemistry. The induction of apoptosis was analyzed by flow cytometry. Results: Our results demonstrate that one-day-old naked mole rats have higher levels of autophagy than one-day-old short-lived C57BL/6 mice, and that both adult naked mole rats (eight months old and adult C57BL/6 mice (eight weeks old have high basal levels of autophagy, which may be an important mechanism inhibiting aging and reducing the risk of age-related diseases. Conclusion: Here, we report that autophagy facilitated the survival of hepatic stellate cells from the naked mole rat, and that treatment with 3-MA or rapamycin increased the ratio of apoptotic cells to normal hepatic stellate cells.

  16. Ulk1-mediated autophagy plays an essential role in mitochondrial remodeling and functional regeneration of skeletal muscle.

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    Call, Jarrod A; Wilson, Rebecca J; Laker, Rhianna C; Zhang, Mei; Kundu, Mondira; Yan, Zhen

    2017-06-01

    Autophagy is a conserved cellular process for degrading aggregate proteins and dysfunctional organelle. It is still debatable if autophagy and mitophagy (a specific process of autophagy of mitochondria) play important roles in myogenic differentiation and functional regeneration of skeletal muscle. We tested the hypothesis that autophagy is critical for functional regeneration of skeletal muscle. We first observed time-dependent increases (3- to 6-fold) of autophagy-related proteins (Atgs), including Ulk1, Beclin1, and LC3, along with reduced p62 expression during C2C12 differentiation, suggesting increased autophagy capacity and flux during myogenic differentiation. We then used cardiotoxin (Ctx) or ischemia-reperfusion (I/R) to induce muscle injury and regeneration and observed increases in Atgs between days 2 and 7 in adult skeletal muscle followed by increased autophagy flux after day 7 Since Ulk1 has been shown to be essential for mitophagy, we asked if Ulk1 is critical for functional regeneration in skeletal muscle. We subjected skeletal muscle-specific Ulk1 knockout mice (MKO) to Ctx or I/R. MKO mice had significantly impaired recovery of muscle strength and mitochondrial protein content post-Ctx or I/R. Imaging analysis showed that MKO mice have significantly attenuated recovery of mitochondrial network at 7 and 14 days post-Ctx. These findings suggest that increased autophagy protein and flux occur during muscle regeneration and Ulk1-mediated mitophagy is critical for recovery for the mitochondrial network and hence functional regeneration. Copyright © 2017 the American Physiological Society.

  17. Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells.

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    Wang, Bin; Chen, Linfeng; Ni, Zhenhong; Dai, Xufang; Qin, Liyan; Wu, Yaran; Li, Xinzhe; Xu, Liang; Lian, Jiqin; He, Fengtian

    2014-11-01

    Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1(Thr163) phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Autophagy induction contributes to GDC-0349 resistance in head and neck squamous cell carcinoma (HNSCC) cells

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    Zhou, Yajuan; Peng, Yi [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan (China); Tang, Hao [Department of Pathology, Hubei Cancer Hospital, Wuhan 430071 (China); He, Xiaojun; Wang, Zhaohua [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan (China); Hu, Desheng, E-mail: hudeshengvvip@sina.com [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan (China); Zhou, Xiaoyi, E-mail: zhouxy1218@126.com [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan (China)

    2016-08-19

    Dysregulation of mammalian target of rapamycin (mTOR) signaling contributes to head and neck squamous cell carcinoma (HNSCC) tumorigenesis and progression. In the current study, we tested the anti-HNSCC cell activity by GDC-0349, a selective ATP-competitive inhibitor of mTOR. We showed that GDC-0349 inhibited proliferation of established and primary human HNSCC cells bearing high-level of p-AKT/p-S6K. Further, it induced caspase-dependent apoptosis in the HNSCC cells. GDC-0349 blocked mTORC1 and mTORC2 activation, yet it simultaneously induced autophagy activation in HNSCC cells. The latter was evidenced by induction of LC3B-II, Beclin-1 and Autophagy-related (ATG)-7, as well as downregulation of p62. Autophagy inhibitors (3-methyladenine and bafilomycin A1) or ATG-7 siRNA dramatically potentiated GDC-0349’s cytotoxicity against HNSCC cells. Intriguingly, we showed that ceramide (C14), a pro-apoptotic sphingolipid, also induced ATG-7 degradation, and sensitized HNSCC cells to GDC-0349. Collectively, the preclinical study provided evidences to support GDC-0349 as a promising anti-HNSCC agent. GDC-0349 sensitization may be achieved via autophagy inhibition. - Highlights: • GDC-0349 inhibits proliferation of HNSCC cells bearing high-level of p-AKT/p-S6K. • GDC-0349 activates caspase-dependent apoptosis in HNSCC cells. • Simultaneous blockage of mTORC1/2 by GDC-0349 induces autophagy activation. • Autophagy inhibitor or ATG-7 siRNA potentiates GDC-0349’s cytotoxicity. • C14 ceramide downregulates ATG-7 and sensitizes HNSCC cells to GDC-0349.

  19. A Dual Role of P53 in Regulating Colistin-Induced Autophagy in PC-12 Cells

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    Ziyin Lu

    2017-10-01

    Full Text Available This study aimed to investigate the mechanism of p53 in regulating colistin-induced autophagy in PC-12 cells. Importantly, cells were treated with 125 μg/ml colistin for 12 and 24 h after transfection with p53 siRNA or recombinant plasmid. The hallmarks of autophagy and apoptosis were examined by real-time PCR and western blot, fluorescence/immunofluorescence microscopy, and electron microscopy. The results showed that silencing of p53 leads to down-regulation of Atg5 and beclin1 for 12 h while up-regulation at 24 h and up-regulation of p62 noted. The ratio of LC3-II/I and autophagic vacuoles were significantly increased at 24 h, but autophagy flux was blocked. The cleavage of caspase3 and PARP (poly ADP-ribose polymerase were enhanced, while PC-12-sip53 cells exposed to 3-MA showed down-regulation of apoptosis. By contrast, the expression of autophagy-related genes and protein reduced in p53 overexpressing cells following a time dependent manner. Meanwhile, there was an increase in the expression of activated caspase3 and PARP, condensed and fragmented nuclei were evident. Conclusively, the data supported that silencing of p53 promotes impaired autophagy, which acts as a pro-apoptotic induction factor in PC-12 cells treated with colistin for 24 h, and overexpression of p53 inhibits autophagy and accelerates apoptosis. Hence, it has been suggested that p53 could not act as a neuro-protective target in colistin-induced neurotoxicity.

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

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    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.

  1. Proteomics Insights into Autophagy.

    Science.gov (United States)

    Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

    2017-10-01

    Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Autophagy in plant pathogenic fungi.

    Science.gov (United States)

    Liu, Xiao-Hong; Xu, Fei; Snyder, John Hugh; Shi, Huan-Bin; Lu, Jian-Ping; Lin, Fu-Cheng

    2016-09-01

    Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. MicroRNA 17-5p regulates autophagy in Mycobacterium tuberculosis-infected macrophages by targeting Mcl-1 and STAT3.

    Science.gov (United States)

    Kumar, Ranjeet; Sahu, Sanjaya Kumar; Kumar, Manish; Jana, Kuladip; Gupta, Pushpa; Gupta, Umesh D; Kundu, Manikuntala; Basu, Joyoti

    2016-05-01

    Autophagy plays a crucial role in the control of bacterial burden during Mycobacterium tuberculosis infection. MicroRNAs (miRNAs) are small non-coding RNAs that regulate immune signalling and inflammation in response to challenge by pathogens. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the role of miRNAs in regulating M. tuberculosis-induced autophagy in macrophages. Here, we demonstrate that M. tuberculosis infection leads to downregulation of miR-17 and concomitant upregulation of its targets Mcl-1 and STAT3, a transcriptional activator of Mcl-1. Forced expression of miR-17 reduces expression of Mcl-1 and STAT3 and also the interaction between Mcl-1 and Beclin-1. This is directly linked to enhanced autophagy, because Mcl-1 overexpression attenuates the effects of miR-17. At the same time, transfection with a kinase-inactive mutant of protein kinase C δ (PKCδ) (an activator of STAT3) augments M. tuberculosis-induced autophagy, and miR-17 overexpression diminishes phosphorylation of PKCδ, suggesting that an miR-17/PKC δ/STAT3 axis regulates autophagy during M. tuberculosis infection. © 2015 John Wiley & Sons Ltd.

  4. Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells.

    Science.gov (United States)

    Kaewpiboon, Chutima; Boonnak, Nawong; Kaowinn, Sirichat; Chung, Young-Hwa

    2018-02-15

    Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4. Copyright © 2017. Published by Elsevier Ltd.

  5. Overexpression of let-7a increases neurotoxicity in a PC12 cell model of Alzheimer's disease via regulating autophagy.

    Science.gov (United States)

    Gu, Huizi; Li, Lan; Cui, Chen; Zhao, Zihui; Song, Guijun

    2017-10-01

    Increased deposition of β-amyloid (Aβ) protein is one of the typical characteristics of Alzheimer's disease (AD). Recent evidence has demonstrated that the microRNA let-7 family, which is highly expressed in the central nervous system, participates in the regulation of pathologic processes of AD. In the present study, the effect of let-7a overexpression on Aβ1-40-induced neurotoxicity was evaluated in PC12 and SK-N-SH cells. The results indicated that overexpression of let-7a enhanced the neurotoxicity induced by Aβ1-40 in PC12 and SK-N-SH cells. In addition, the apoptosis induced by Aβ1-40 in PC12 and SK-N-SH cells was increased by let-7a overexpression. Furthermore, Aβ1-40 treatment increased the protein levels of microtubule-associated protein 1A/1B-light chain 3 (LC3) and beclin-1 and increased the LC3 II/I ratio. The mRNA expression levels of beclin-1, autophagy protein 5 (Atg-5) and Atg-7 were also increased by Aβ1-40 treatment in PC12 cells. Let-7a overexpression further upregulated the above autophagy-related markers. Furthermore, the protein level of p62 was increased by Aβ1-40 treatment, and this was further enhanced by let-7a overexpression. Finally, the present results demonstrated that the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was involved in the autophagy regulation by let-7a. In conclusion, the present study demonstrates that the neurotoxicity induced by Aβ1-40 is augmented by let-7a overexpression via regulation of autophagy, and the PI3K/Akt/mTOR signaling pathway also serves a function in this process.

  6. L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress, Autophagy, and Apoptosis.

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    Huang, Ya-Ni; Yang, Ling-Yu; Wang, Jing-Ya; Lai, Chien-Cheng; Chiu, Chien-Tsai; Wang, Jia-Yi

    2017-01-01

    Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.

  7. Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells

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    Lin JF

    2017-05-01

    cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1 was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis.Conclusion: Collectively, the study results indicated that cisplatin-induced autophagy is mediated by BECN1 in BC cells. Therefore, combinative treatment using cisplatin and autophagy inhibitors could potentially overcome cisplatin resistance related to autophagy induction. Keywords: autophagy inhibition, autophagy related genes, apoptosis, cisplatin resistance, human urinary bladder urothelial carcinoma, lentiviral-based shRNA

  8. Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

    Science.gov (United States)

    Zhang, Jun-Xia; Qu, Xin-Liang; Chu, Peng; Xie, Du-Jiang; Zhu, Lin-Lin; Chao, Yue-Lin; Li, Li; Zhang, Jun-Jie; Chen, Shao-Liang

    2018-05-01

    Uncoupled endothelial nitric oxide synthase (eNOS) produces O 2 - instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O 2 - production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O 2 - burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O 2 - releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Melatonin reverses H2 O2 -induced senescence in SH-SY5Y cells by enhancing autophagy via sirtuin 1 deacetylation of the RelA/p65 subunit of NF-κB.

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    Nopparat, Chutikorn; Sinjanakhom, Puritat; Govitrapong, Piyarat

    2017-08-01

    Autophagy, a degradation mechanism that plays a major role in maintaining cellular homeostasis and diminishes in aging, is considered an aging characteristic. Melatonin is an important hormone that plays a wide range of physiological functions, including the anti-aging effect, potentially via the regulation of the Sirtuin1 (SIRT1) pathway. The deacetylation ability of SIRT1 is important for controlling the function of several transcription factors, including nuclear factor kappa B (NF-ĸB). Apart from inflammation, NF-ĸB can regulate autophagy by inhibiting Beclin1, an initiator of autophagy. Although numerous studies have revealed the role of melatonin in regulating autophagy, very limited experiments have shown that melatonin can increase autophagic activity via SIRT1 in a senescent model. This study focuses on the effect of melatonin on autophagy via the deacetylation activity of SIRT1 on RelA/p65, a subunit of NF-ĸB, to determine whether melatonin can attenuate the aging condition. SH-SY5Y cells were treated with H 2 O 2 to induce the senescent state. These results demonstrated that melatonin reduced a number of beta-galactosidase (SA-βgal)-positive cells, a senescent marker. In addition, melatonin increased the protein levels of SIRT1, Beclin1, and LC3-II, a hallmark protein of autophagy, and reduced the levels of acetylated-Lys310 in the p65 subunit of NF-ĸB in SH-SY5Y cells treated with H 2 O 2 . Furthermore, in the presence of SIRT1 inhibitor, melatonin failed to increase autophagic markers. The present data indicate that melatonin enhances autophagic activity via the SIRT1 signaling pathway. Taken together, we propose that in modulating autophagy, melatonin may provide a therapeutically beneficial role in the anti-aging processes. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Kim, In-Ryoung; Kim, Seong-Eon; Baek, Hyun-Su; Kim, Bok-Joo; Kim, Chul-Hoon; Chung, In-Kyo; Park, Bong-Soo; Shin, Sang-Hun

    2016-08-31

    beclin-1, SQSTM1/p62 and the conversion of LC3-II from LC3-I. The application of 3-MA decreased the activity of ALP and the autophagy induced by kaempferol. In the RT-PCR analysis, the expression of RUNX-2, osterix, BMP-2 and collagen I was decreased. The present study showed that kaempferol stimulated the osteogenic differentiation of cultured osteoblasts by inducing autophagy.

  11. Neferine augments therapeutic efficacy of cisplatin through ROS- mediated non-canonical autophagy in human lung adenocarcinoma (A549 cells).

    Science.gov (United States)

    Kalai Selvi, Sivalingam; Vinoth, Amirthalingam; Varadharajan, Thiyagarajan; Weng, Ching Feng; Vijaya Padma, Viswanadha

    2017-05-01

    Combination of dietary components with chemotherapy drugs is an emerging new strategy for cancer therapy to increase antitumor responses. Neferine, major bisbenzylisoquinoline alkaloid isolated from the seed embryo of Nelumbo nucifera (Lotus). In the present study, we investigated the efficacy of the combinatorial regimen of neferine and cisplatin compared to cisplatin high dose in human lung adenocarcinoma (A549) cells. Co-treatment with neferine enhanced cisplatin-induced autophagy in A549 cells was accompanied by Acidic vesicular accumulation (AVO), enhanced generation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH), down regulation of PI3K/AKT/mTOR pathway, conversion of LC3B-I to LC3B-II. This enhanced autophagy developed via a non-canonical mechanism that did not require Beclin-1, PI3KCIII. In conclusion, these results suggest that neferine enhances cisplatin -induced autophagic cancer cell death through downregulation of PI3K/Akt/mTOR signaling pro-survival pathway and ROS- mediated Beclin-1 and PI3K CIII independent autophagy in human lung adenocarcinoma (A549 cells). Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Dopamine Oxidation and Autophagy

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    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.

  13. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    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.

  14. Autophagy in Measles Virus Infection

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    Aurore Rozières

    2017-11-01

    Full Text Available Autophagy is a biological process that helps cells to recycle obsolete cellular components and which greatly contributes to maintaining cellular integrity in response to environmental stress factors. Autophagy is also among the first lines of cellular defense against invading microorganisms, including viruses. The autophagic destruction of invading pathogens, a process referred to as xenophagy, involves cytosolic autophagy receptors, such as p62/SQSTM1 (Sequestosome 1 or NDP52/CALCOCO2 (Nuclear Dot 52 KDa Protein/Calcium Binding And Coiled-Coil Domain 2, which bind to microbial components and target them towards growing autophagosomes for degradation. However, most, if not all, infectious viruses have evolved molecular tricks to escape from xenophagy. Many viruses even use autophagy, part of the autophagy pathway or some autophagy-associated proteins, to improve their infectious potential. In this regard, the measles virus, responsible for epidemic measles, has a unique interface with autophagy as the virus can induce multiple rounds of autophagy in the course of infection. These successive waves of autophagy result from distinct molecular pathways and seem associated with anti- and/or pro-measles virus consequences. In this review, we describe what the autophagy–measles virus interplay has taught us about both the biology of the virus and the mechanistic orchestration of autophagy.

  15. Autophagy in C. elegans development.

    Science.gov (United States)

    Palmisano, Nicholas J; Meléndez, Alicia

    2018-04-27

    Autophagy involves the sequestration of cytoplasmic contents in a double-membrane structure referred to as the autophagosome and the degradation of its contents upon delivery to lysosomes. Autophagy activity has a role in multiple biological processes during the development of the nematode Caenorhabditis elegans. Basal levels of autophagy are required to remove aggregate prone proteins, paternal mitochondria, and spermatid-specific membranous organelles. During larval development, autophagy is required for the remodeling that occurs during dauer development, and autophagy can selectively degrade components of the miRNA-induced silencing complex, and modulate miRNA-mediated silencing. Basal levels of autophagy are important in synapse formation and in the germ line, to promote the proliferation of proliferating stem cells. Autophagy activity is also required for the efficient removal of apoptotic cell corpses by promoting phagosome maturation. Finally, autophagy is also involved in lipid homeostasis and in the aging process. In this review, we first describe the molecular complexes involved in the process of autophagy, its regulation, and mechanisms for cargo recognition. In the second section, we discuss the developmental contexts where autophagy has been shown to be important. Studies in C. elegans provide valuable insights into the physiological relevance of this process during metazoan development. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy

    Science.gov (United States)

    Yin, Fei; Meng, Chunyang; Lu, Rifeng; Li, Lei; Zhang, Ying; Chen, Hao; Qin, Yonggang; Guo, Li

    2014-01-01

    Bone marrow mesenchymal stem cells can differentiate into neurons and astrocytes after transplantation in the spinal cord of rats with ischemia/reperfusion injury. Although bone marrow mesenchymal stem cells are known to protect against spinal cord ischemia/reperfusion injury through anti-apoptotic effects, the precise mechanisms remain unclear. In the present study, bone marrow mesenchymal stem cells were cultured and proliferated, then transplanted into rats with ischemia/reperfusion injury via retro-orbital injection. Immunohistochemistry and immunofluorescence with subsequent quantification revealed that the expression of the axonal regeneration marker, growth associated protein-43, and the neuronal marker, microtubule-associated protein 2, significantly increased in rats with bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Furthermore, the expression of the autophagy marker, microtubule-associated protein light chain 3B, and Beclin 1, was significantly reduced in rats with the bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Western blot analysis showed that the expression of growth associated protein-43 and neurofilament-H increased but light chain 3B and Beclin 1 decreased in rats with the bone marrow mesenchymal stem cell transplantation. Our results therefore suggest that bone marrow mesenchymal stem cell transplantation promotes neurite growth and regeneration and prevents autophagy. These responses may likely be mechanisms underlying the protective effect of bone marrow mesenchymal stem cells against spinal cord ischemia/reperfusion injury. PMID:25374587

  17. Emblica officinalis extract induces autophagy and inhibits human ovarian cancer cell proliferation, angiogenesis, growth of mouse xenograft tumors.

    Directory of Open Access Journals (Sweden)

    Alok De

    Full Text Available Patients with ovarian cancer (OC may be treated with surgery, chemotherapy and/or radiation therapy, although none of these strategies are very effective. Several plant-based natural products/dietary supplements, including extracts from Emblicaofficinalis (Amla, have demonstrated potent anti-neoplastic properties. In this study we determined that Amla extract (AE has anti-proliferative effects on OC cells under both in vitro and in vivo conditions. We also determined the anti-proliferative effects one of the components of AE, quercetin, on OC cells under in vitro conditions. AE did not induce apoptotic cell death, but did significantly increase the expression of the autophagic proteins beclin1 and LC3B-II under in vitro conditions. Quercetin also increased the expression of the autophagic proteins beclin1 and LC3B-II under in vitro conditions. AE also significantly reduced the expression of several angiogenic genes, including hypoxia-inducible factor 1α (HIF-1α in OVCAR3 cells. AE acted synergistically with cisplatin to reduce cell proliferation and increase expression of the autophagic proteins beclin1 and LC3B-II under in vitro conditions. AE also had anti-proliferative effects and induced the expression of the autophagic proteins beclin1 and LC3B-II in mouse xenograft tumors. Additionally, AE reduced endothelial cell antigen - CD31 positive blood vessels and HIF-1α expression in mouse xenograft tumors. Together, these studies indicate that AE inhibits OC cell growth both in vitro and in vivo possibly via inhibition of angiogenesis and activation of autophagy in OC. Thus AE may prove useful as an alternative or adjunct therapeutic approach in helping to fight OC.

  18. C-myb Regulates Autophagy for Pulp Vitality in Glucose Oxidative Stress.

    Science.gov (United States)

    Lee, Y H; Kim, H S; Kim, J S; Yu, M K; Cho, S D; Jeon, J G; Yi, H K

    2016-04-01

    Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B-light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases. © International & American Associations

  19. Excessive apoptosis and defective autophagy contribute to developmental testicular toxicity induced by fluoride.

    Science.gov (United States)

    Zhang, Shun; Niu, Qiang; Gao, Hui; Ma, Rulin; Lei, Rongrong; Zhang, Cheng; Xia, Tao; Li, Pei; Xu, Chunyan; Wang, Chao; Chen, Jingwen; Dong, Lixing; Zhao, Qian; Wang, Aiguo

    2016-05-01

    Fluoride, a ubiquitous environmental contaminant, is known to impair testicular functions and fertility; however the underlying mechanisms remain obscure. In this study, we used a rat model to mimic human exposure and sought to investigate the roles of apoptosis and autophagy in testicular toxicity of fluoride. Sprague-Dawley rats were developmentally exposed to 25, 50, or 100 mg/L sodium fluoride (NaF) via drinking water from pre-pregnancy to post-puberty, and then the testes of offspring were excised on postnatal day 56. Our results demonstrated that developmental NaF exposure induced an enhanced testicular apoptosis, as manifested by a series of hallmarks such as caspase-3 activation, chromatin condensation and DNA fragmentation. Further study revealed that fluoride exposure elicited significant elevations in the levels of cell surface death receptor Fas with a parallel increase in cytoplasmic cytochrome c, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. Intriguingly, fluoride treatment also simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II but not Beclin1. Unexpectedly, the expression of p62, a substrate that is degraded by autophagy, was also significantly elevated, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation rather than increased formation. Importantly, these were associated with marked histopathological lesions including spermatogenic failure and germ cell loss, along with severe ultrastructural abnormalities in testes. Taken together, our findings provide deeper insights into roles of excessive apoptosis and defective autophagy in the aggravation of testicular damage, which could contribute to a better understanding of fluoride-induced male reproductive toxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

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    Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  1. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    International Nuclear Information System (INIS)

    Tashiro, Kanae; Shishido, Mayumi; Fujimoto, Keiko; Hirota, Yuko; Yo, Kazuyuki; Gomi, Takamasa; Tanaka, Yoshitaka

    2014-01-01

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility

  2. The roles of autophagy and hypoxia in human inflammatory periapical lesions.

    Science.gov (United States)

    Huang, H Y; Wang, W C; Lin, P Y; Huang, C P; Chen, C Y; Chen, Y K

    2018-02-01

    To determine the expressions of hypoxia-related [hypoxia-inducible transcription factors (HIF)-1α, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and phospho-adenosine monophosphate activated protein kinase (pAMPK)] and autophagy-related [microtubule-associated protein 1 light chain 3 (LC3), beclin-1 (BECN-1), autophagy-related gene (Atg)5-12, and p62] proteins in human inflammatory periapical lesions. Fifteen samples of radicular cysts (RCs) and 21 periapical granulomas (PGs), combined with 17 healthy dental pulp tissues, were examined. Enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β cytokine; immunohistochemical (IHC) and Western blot (WB) analyses were employed to examine autophagy-related and hypoxia-related proteins. Transmission electron microscopy (TEM) was used to explore the ultrastructural morphology of autophagy in periapical lesions. Nonparametric Kruskal-Wallis tests and Mann-Whitney U-tests were used for statistical analyses. ELISA revealed a significantly higher (P periapical lesions than in normal pulp tissue. Immunoscores of IHC expressions of pAMPK, HIF-1α, BNIP3, BECN-1 and Atg5-12 proteins in periapical lesions were significantly higher (P periapical lesions were noted as compared to normal pulp tissue. Upon TEM, ultrastructural double-membrane autophagosomes and autolysosomes were observed in PGs and RCs. Autophagy associated with hypoxia may play a potential causative role in the development and maintenance of inflamed periapical lesions. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  3. Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts.

    Science.gov (United States)

    Guo, Rui; Hu, Nan; Kandadi, Machender R; Ren, Jun

    2012-04-01

    Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

  4. Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway.

    Science.gov (United States)

    Hu, Junzheng; Cui, Weiding; Ding, Wenxiao; Gu, Yanqing; Wang, Zhen; Fan, Weimin

    2017-01-01

    Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN), secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H2O2-induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes. H2O2 was used to induce apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H2O2. Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosis-related proteins were identified by Western blot analysis. H2O2 (400 µM)-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 µg/mL). gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H2O2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H2O2. Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role in the induction of autophagy and protection of H2O2-induced chondrocytes apoptosis by gAPN. g

  5. Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway

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    Junzheng Hu

    2017-08-01

    Full Text Available Background/Aims: Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN, secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H2O2-induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes. Methods: H2O2 was used to induce apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H2O2. Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosis-related proteins were identified by Western blot analysis. Results: H2O2 (400 µM-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 µg/mL. gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H2O2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H2O2. Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role in the induction of autophagy and protection of

  6. Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up.

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    Arnaud François

    Full Text Available Recent findings indicate that microglia in Alzheimer's disease (AD is senescent whereas peripheral blood mononuclear cells (PBMCs could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1-42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA, and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3 were quantified by western blot and cytokines (Interleukin (IL-1β, Tumor necrosis Factor (TNF-α and IL-6 by Luminex X-MAP® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.

  7. Stat3 Expression and Its Correlation with Proliferation and Apoptosis/Autophagy in Gliomas

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    Valentina Caldera

    2008-01-01

    Full Text Available Signal transducer and activator of transcription-3 (Stat3 was studied along with several steps of the PI3/Akt pathway in a series of 64 gliomas that included both malignant and low-grade tumors, using quantitative immunohistochemistry, Western blotting, and molecular biology techniques. The goal of the study was to investigate whether activated Stat3 (phospho-Stat3 levels correlated with cell proliferation, apoptosis, and autophagy. Stat3 and activated Akt (phospho-Akt expression increased with malignancy grade, but did not correlate with proliferation and survival within the category of glioblastomas. A correlation of Stat3 with Akt was found, indicating a regulation of the former by the PI3/Akt pathway, which, in turn, was in relation with EGFR amplification. Stat3 and Akt did not show any correlation with apoptosis, whereas they showed an inverse correlation with Beclin 1, a stimulator of autophagy, which was rarely positive in glioblastomas. Autophagy seems then to be inactivated in malignant gliomas.

  8. Autophagy in retinal ganglion cells in a rhesus monkey chronic hypertensive glaucoma model.

    Directory of Open Access Journals (Sweden)

    Shuifeng Deng

    Full Text Available Primary open angle glaucoma (POAG is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs. In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi and late or degraded autophagic vacuoles (AVd accumulated in the ganglion cell layer (GCL and in the inner plexiform layer (IPL as determined by transmission electron microscopy (TEM analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1 and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.

  9. Excessive apoptosis and defective autophagy contribute to developmental testicular toxicity induced by fluoride

    International Nuclear Information System (INIS)

    Zhang, Shun; Niu, Qiang; Gao, Hui; Ma, Rulin; Lei, Rongrong; Zhang, Cheng; Xia, Tao; Li, Pei; Xu, Chunyan; Wang, Chao; Chen, Jingwen; Dong, Lixing; Zhao, Qian; Wang, Aiguo

    2016-01-01

    Fluoride, a ubiquitous environmental contaminant, is known to impair testicular functions and fertility; however the underlying mechanisms remain obscure. In this study, we used a rat model to mimic human exposure and sought to investigate the roles of apoptosis and autophagy in testicular toxicity of fluoride. Sprague–Dawley rats were developmentally exposed to 25, 50, or 100 mg/L sodium fluoride (NaF) via drinking water from pre-pregnancy to post-puberty, and then the testes of offspring were excised on postnatal day 56. Our results demonstrated that developmental NaF exposure induced an enhanced testicular apoptosis, as manifested by a series of hallmarks such as caspase-3 activation, chromatin condensation and DNA fragmentation. Further study revealed that fluoride exposure elicited significant elevations in the levels of cell surface death receptor Fas with a parallel increase in cytoplasmic cytochrome c, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. Intriguingly, fluoride treatment also simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II but not Beclin1. Unexpectedly, the expression of p62, a substrate that is degraded by autophagy, was also significantly elevated, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation rather than increased formation. Importantly, these were associated with marked histopathological lesions including spermatogenic failure and germ cell loss, along with severe ultrastructural abnormalities in testes. Taken together, our findings provide deeper insights into roles of excessive apoptosis and defective autophagy in the aggravation of testicular damage, which could contribute to a better understanding of fluoride-induced male reproductive toxicity. - Highlights: • Rats were developmentally exposed to fluoride from pre-pregnancy to post-puberty. • Both excessive apoptosis and defective autophagy are involved in

  10. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

    International Nuclear Information System (INIS)

    Xia, D.Y.; Li, W.; Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K.

    2013-01-01

    Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis

  11. Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Xia, D.Y. [Department of Neurology, Navy General Hospital of PLA, Beijing (China); Li, W. [General Hospital of Shenyang Military Command, Department of Neurology, Shenyang, China, Department of Neurology, General Hospital of Shenyang Military Command, Shenyang (China); Qian, H.R.; Yao, S.; Liu, J.G.; Qi, X.K. [Department of Neurology, Navy General Hospital of PLA, Beijing (China)

    2013-08-10

    Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

  12. Conformational flexibility of BECN1: Essential to its key role in autophagy and beyond: BECN1 Structure and Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Mei, Yang [Department of Chemistry and Biochemistry, North Dakota State University, Fargo North Dakota 58108-6050; Glover, Karen [Department of Chemistry and Biochemistry, North Dakota State University, Fargo North Dakota 58108-6050; Su, Minfei [Department of Chemistry and Biochemistry, North Dakota State University, Fargo North Dakota 58108-6050; Sinha, Sangita C. [Department of Chemistry and Biochemistry, North Dakota State University, Fargo North Dakota 58108-6050

    2016-08-13

    BECN1 (Beclin 1), a highly conserved eukaryotic protein, is a key regulator of autophagy, a cellular homeostasis pathway, and also participates in vacuolar protein sorting, endocytic trafficking, and apoptosis. BECN1 is important for embryonic development, the innate immune response, tumor suppression, and protection against neurodegenerative disorders, diabetes, and heart disease. BECN1 mediates autophagy as a core component of the class III phosphatidylinositol 3-kinase complexes. However, the exact mechanism by which it regulates the activity of these complexes, or mediates its other diverse functions is unclear. BECN1 interacts with several diverse protein partners, perhaps serving as a scaffold or interaction hub for autophagy. Based on extensive structural, biophysical and bioinformatics analyses, BECN1 consists of an intrinsically disordered region (IDR), which includes a BH3 homology domain (BH3D); a flexible helical domain (FHD); a coiled-coil domain (CCD); and a β-α-repeated autophagy-specific domain (BARAD). Each of these BECN1 domains mediates multiple diverse interactions that involve concomitant conformational changes. Thus, BECN1 conformational flexibility likely plays a key role in facilitating diverse protein interactions. Further, BECN1 conformation and interactions are also modulated by numerous post-translational modifications. A better structure-based understanding of the interplay between different BECN1 conformational and binding states, and the impact of post-translational modifications will be essential to elucidating the mechanism of its multiple biological roles.

  13. SIRT1 protects cardiac cells against apoptosis induced by zearalenone or its metabolites α- and β-zearalenol through an autophagy-dependent pathway

    International Nuclear Information System (INIS)

    Ben Salem, Intidhar; Boussabbeh, Manel; Da Silva, Julie Pires; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2017-01-01

    Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). In the present study, we investigated the underlying mechanism of the toxicity induced by ZEN, α-ZOL and β-ZOL in cardiac cells (H9c2). We show that treatment with ZEN or its metabolites induces the activation of the mitochondrial pathway of apoptosis as characterized by an increase in ROS generation, a loss of mitochondrial transmembrane potential (ΔΨm) and an activation of caspases. Besides, we demonstrate that these mycotoxins promote the activation of autophagy before the onset of apoptosis. Indeed, we observed that a short-time (6 h) treatment with ZEN, α-ZOL or β-ZOL, increased the level of Beclin-1 and LC3-II and induced the accumulation of the CytoID® autophagy detection probe. Moreover, the inhibition of autophagy by Chloroquine significantly increased cell death induced by ZEN, α-ZOL or β-ZOL, suggesting that the activation of autophagy serves as a cardioprotective mechanism against these mycotoxins. In addition, we found that the inhibition (EX527) or the knockdown of SIRT1 (siRNA) significantly increased apoptosis induced by ZEN or its derivatives, whereas SIRT1 activation with RSV greatly prevents the cytotoxic effects of these mycotoxins. By contrast, when autophagy was inhibited by CQ, the activation of SIRT1 by RSV had no protection against the cardiotoxicity of ZEN or its metabolites, suggesting that SIRT1 protects cardiac cells by an autophagy-dependent pathway. - Highlights: • ZEN, α- and β-ZOL induce the mitochondrial pathway of apoptosis in cardiac cells. • Inhibition of autophagy enhanced ZEN-, α-ZOL- and β-ZOL-induced apoptosis. • SIRT1 activates autophagy to protect cells from ZEN, α- and β-ZOL-induced toxicity.

  14. Heme oxygenase-1-mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium-treated mice

    Science.gov (United States)

    Surolia, Ranu; Karki, Suman; Kim, Hyunki; Yu, Zhihong; Kulkarni, Tejaswini; Mirov, Sergey B.; Carter, A. Brent; Rowe, Steven M.; Matalon, Sadis; Thannickal, Victor J.; Agarwal, Anupam

    2015-01-01

    Pulmonary exposure to cadmium, a major component of cigarette smoke, has a dramatic impact on lung function and the development of emphysema. Cigarette smoke exposure induces heme oxygenase-1 (HO-1), a cytoprotective enzyme. In this study, we employed a truncated mouse model of emphysema by intratracheal instillation of cadmium (CdCl2) solution (0.025% per 1 mg/kg body wt) in HO-1+/+, HO-1−/−, and overexpressing humanized HO-1 bacterial artificial chromosome (hHO-1BAC) mice. We evaluated the role of HO-1 in cadmium-induced emphysema in mice by analyzing histopathology, micro-computed tomography scans, and lung function tests. CdCl2-exposed HO-1−/− mice exhibited more severe emphysema compared with HO-1+/+ or hHO-1BAC mice. Loss of pulmonary endothelial cells (PECs) from the alveolar capillary membrane is recognized to be a target in emphysema. PECs from HO-1+/+, HO-1−/−, and hHO-1BAC were employed to define the underlying molecular mechanism for the protection from emphysema by HO-1. Electron microscopy, expression of autophagic markers (microtubule-associated protein 1B-light chain 3 II, autophagy protein 5, and Beclin1) and apoptotic marker (cleaved caspase 3) suggested induction of autophagy and apoptosis in PECs after CdCl2 treatment. CdCl2-treated HO-1−/− PECs exhibited downregulation of autophagic markers and significantly increased cleaved caspase 3 expression and activity (∼4-fold higher). Moreover, hHO-1BAC PECs demonstrated upregulated autophagy and absence of cleaved caspase 3 expression or activity. Pretreatment of HO-1+/+ PECs with rapamycin induced autophagy and resulted in reduced cell death upon cadmium treatment. Induction of autophagy following CdCl2 treatment was found to be protective from apoptotic cell death. HO-1 induced protective autophagy in PECs and mitigated cadmium-induced emphysema. PMID:26071551

  15. Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice

    Science.gov (United States)

    Huang, Yalan; Feng, Yanhai; Wang, Yu; Wang, Pei; Wang, Fengjun; Ren, Hui

    2018-01-01

    The disruption of intestinal barrier plays a vital role in the pathophysiological changes after severe burn injury, however, the underlying mechanisms are poorly understood. Severe burn causes the disruption of intestinal tight junction (TJ) barrier. Previous studies have shown that endoplasmic reticulum (ER) stress and autophagy are closely associated with the impairment of intestinal mucosa. Thus, we hypothesize that ER stress and autophagy are likely involved in burn injury-induced intestinal epithelial barrier dysfunction. Mice received a 30% total body surface area (TBSA) full-thickness burn, and were sacrificed at 0, 1, 2, 6, 12 and 24 h postburn. The results showed that intestinal permeability was increased significantly after burn injury, accompanied by the damage of mucosa and the alteration of TJ proteins. Severe burn induced ER stress, as indicated by increased intraluminal chaperone binding protein (BIP), CCAAT/enhancer-binding protein homologous protein (CHOP) and inositol-requiring enzyme 1(IRE1)/X-box binding protein 1 splicing (XBP1). Autophagy was activated after burn injury, as evidenced by the increase of autophagy related protein 5 (ATG5), Beclin 1 and LC3II/LC3I ratio and the decrease of p62. Besides, the number of autophagosomes was also increased after burn injury. The levels of p-PI3K(Ser191), p-PI3K(Ser262), p-AKT(Ser473), and p-mTOR were decreased postburn, suggesting that autophagy-related PI3K/AKT/mTOR pathway is involved in the intestinal epithelial barrier dysfunction following severe burn. In summary, severe burn injury induces the ER stress and autophagy in intestinal epithelia, leading to the disruption of intestinal barrier. PMID:29740349

  16. Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes

    Directory of Open Access Journals (Sweden)

    Yu-Miao Wei

    2013-06-01

    Full Text Available Background/Aims: In addition to their action of lowering blood cholesterol levels, statins modulate biological characteristics and functions of arterial myocytes such as viability, proliferation, apoptosis, survival and contraction. The present study tested whether simvastatin, as a prototype statin, enhances autophagy in coronary arterial myocytes (CAMs to thereby exert their beneficial effects in atherosclerosis. Methods and Results: Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries. Conclusion: Our results indicate that simvastatin inhibits the Rac1-mTOR pathway and thereby increases autophagy in CAMs which may stabilize CAMs in the contractile phenotype to prevent proliferation and growth

  17. Heme oxygenase-1-mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium-treated mice.

    Science.gov (United States)

    Surolia, Ranu; Karki, Suman; Kim, Hyunki; Yu, Zhihong; Kulkarni, Tejaswini; Mirov, Sergey B; Carter, A Brent; Rowe, Steven M; Matalon, Sadis; Thannickal, Victor J; Agarwal, Anupam; Antony, Veena B

    2015-08-01

    Pulmonary exposure to cadmium, a major component of cigarette smoke, has a dramatic impact on lung function and the development of emphysema. Cigarette smoke exposure induces heme oxygenase-1 (HO-1), a cytoprotective enzyme. In this study, we employed a truncated mouse model of emphysema by intratracheal instillation of cadmium (CdCl2) solution (0.025% per 1 mg/kg body wt) in HO-1(+/+), HO-1(-/-), and overexpressing humanized HO-1 bacterial artificial chromosome (hHO-1BAC) mice. We evaluated the role of HO-1 in cadmium-induced emphysema in mice by analyzing histopathology, micro-computed tomography scans, and lung function tests. CdCl2-exposed HO-1(-/-) mice exhibited more severe emphysema compared with HO-1(+/+) or hHO-1BAC mice. Loss of pulmonary endothelial cells (PECs) from the alveolar capillary membrane is recognized to be a target in emphysema. PECs from HO-1(+/+), HO-1(-/-), and hHO-1BAC were employed to define the underlying molecular mechanism for the protection from emphysema by HO-1. Electron microscopy, expression of autophagic markers (microtubule-associated protein 1B-light chain 3 II, autophagy protein 5, and Beclin1) and apoptotic marker (cleaved caspase 3) suggested induction of autophagy and apoptosis in PECs after CdCl2 treatment. CdCl2-treated HO-1(-/-) PECs exhibited downregulation of autophagic markers and significantly increased cleaved caspase 3 expression and activity (∼4-fold higher). Moreover, hHO-1BAC PECs demonstrated upregulated autophagy and absence of cleaved caspase 3 expression or activity. Pretreatment of HO-1(+/+) PECs with rapamycin induced autophagy and resulted in reduced cell death upon cadmium treatment. Induction of autophagy following CdCl2 treatment was found to be protective from apoptotic cell death. HO-1 induced protective autophagy in PECs and mitigated cadmium-induced emphysema. Copyright © 2015 the American Physiological Society.

  18. Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.

    Directory of Open Access Journals (Sweden)

    Jiateng Zhong

    Full Text Available CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER, nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.

  19. Apolipoprotein E-Mimetic Peptide COG1410 Promotes Autophagy by Phosphorylating GSK-3β in Early Brain Injury Following Experimental Subarachnoid Hemorrhage

    Directory of Open Access Journals (Sweden)

    Xinshen Li

    2018-03-01

    Full Text Available COG1410, a mimetic peptide derived from the apolipoprotein E (apoE receptor binding region, exerts positive effect on neurological deficits in early brain injury (EBI after experimental subarachnoid hemorrhage (SAH. Currently the neuroprotective effect of COG1410 includes inhibiting BBB disruption, reducing neuronal apoptosis, and neuroinflammation. However, the effect and mechanism of COG1410 to subcellular organelles disorder have not been fully investigated. As the main pathway for recycling long-lived proteins and damaged organelles, neuronal autophagy is activated in SAH and exhibits neuroprotective effects by reducing the insults of EBI. Pharmacologically elevated autophagy usually contributes to alleviated brain injury, while few of the agents achieved clinical transformation. In this study, we explored the activation of autophagy during EBI by measuring the Beclin-1 and LC3B-II protein levels. Administration of COG1410 notably elevated the autophagic markers expression in neurons, simultaneously reversed the neurological deficits. Furthermore, the up-regulated autophagy by COG1410 was further promoted by p-GSK-3β agonist, whereas decreased by p-GSK-3β inhibitor. Taken together, these data suggest that the COG1410 might be a promising therapeutic strategy for EBI via promoting autophagy in SAH.

  20. Ghrelin Attenuates Retinal Neuronal Autophagy and Apoptosis in an Experimental Rat Glaucoma Model.

    Science.gov (United States)

    Zhu, Ke; Zhang, Meng-Lu; Liu, Shu-Ting; Li, Xue-Yan; Zhong, Shu-Min; Li, Fang; Xu, Ge-Zhi; Wang, Zhongfeng; Miao, Yanying

    2017-12-01

    Ghrelin, a natural ligand for the growth hormone secretagogue receptor type 1a (GHSR-1a), may protect retinal neurons against glaucomatous injury. We therefore characterized the underlying mechanism of the ghrelin/GHSR-1a-mediated neuroprotection with a rat chronic intraocular hypertension (COH) model. The rat COH model was produced by blocking episcleral veins. A combination of immunohistochemistry, Western blot, TUNEL assay, and retrograde labeling of retinal ganglion cells (RGCs) was used. Elevation of intraocular pressure induced a significant increase in ghrelin and GHSR-1a expression in retinal cells, including RGCs and Müller cells. Western blot confirmed that the protein levels of ghrelin exhibited a transient upregulation at week 2 after surgery (G2w), while the GHSR-1a protein levels were maintained at high levels from G2w to G4w. In COH retinas, the ratio of LC3-II/LC-I and beclin1, two autophagy-related proteins, were increased from G1w to G4w, and the cleavage product of caspase3, an apoptotic executioner, was detected from G2w to G4w. Intraperitoneal injection of ghrelin significantly increased the number of surviving RGCs; inhibited the changes of LC3-II/LC-I, beclin1, and the cleavage products of caspase3; and reduced the number of TUNEL-positive cells in COH retinas. Ghrelin treatment also reversed the decreased levels of p-Akt and p-mTOR, upregulated GHSR-1a protein levels, and attenuated glial fibrillary acidic protein levels in COH retinas. All these results suggest that ghrelin may provide neuroprotective effect in COH retinas through activating ghrelin/GHSR-1a system, which was mediated by inhibiting retinal autophagy, ganglion cell apoptosis, and Müller cell gliosis.

  1. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    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

  2. 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.

  3. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    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. PMID:24212825

  4. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available 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 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.

  5. Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion.

    Directory of Open Access Journals (Sweden)

    Yeon Ja Choi

    Full Text Available Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time-dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel

  6. Magnetic ferroferric oxide nanoparticles induce vascular endothelial cell dysfunction and inflammation by disturbing autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lu, E-mail: chaperones@163.com [College of Bioengineering, Henan University of Technology, Lianhua Street, Zhengzhou 450001 (China); Wang, XueQin; Miao, YiMing; Chen, ZhiQiang; Qiang, PengFei; Cui, LiuQing; Jing, Hongjuan [College of Bioengineering, Henan University of Technology, Lianhua Street, Zhengzhou 450001 (China); Guo, YuQi [Department of Obstetrics and Gynecology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450052 (China)

    2016-03-05

    Highlights: • B-Fe{sub 3}O{sub 4}NPs did not induce cell apoptosis or necrosis in HUVECs within 24 h. • B-Fe{sub 3}O{sub 4}NPs induced HUVEC dysfunction and inflammation. • B-Fe{sub 3}O{sub 4}NPs induced enhanced autophagic activity and blockade of autophagy flux. • Suppression of autophagy dysfunction attenuated B-Fe{sub 3}O{sub 4}NP-induced HUVEC dysfunction. - Abstract: Despite the considerable use of magnetic ferroferric oxide nanoparticles (Fe{sub 3}O{sub 4}NPs) worldwide, their safety is still an important topic of debate. In the present study, we detected the toxicity and biological behavior of bare-Fe{sub 3}O{sub 4}NPs (B-Fe{sub 3}O{sub 4}NPs) on human umbilical vascular endothelial cells (HUVECs). Our results showed that B-Fe{sub 3}O{sub 4}NPs did not induce cell death within 24 h even at concentrations up to 400 μg/ml. The level of nitric oxide (NO) and the activity of endothelial NO synthase (eNOS) were decreased after exposure to B-Fe{sub 3}O{sub 4}NPs, whereas the levels of proinflammatory cytokines were elevated. Importantly, B-Fe{sub 3}O{sub 4}NPs increased the accumulation of autophagosomes and LC3-II in HUVECs through both autophagy induction and the blockade of autophagy flux. The levels of Beclin 1 and VPS34, but not phosphorylated mTOR, were increased in the B-Fe{sub 3}O{sub 4}NP-treated HUVECs. Suppression of autophagy induction or stimulation of autophagy flux, at least partially, attenuated the B-Fe{sub 3}O{sub 4}NP-induced HUVEC dysfunction. Additionally, enhanced autophagic activity might be linked to the B-Fe{sub 3}O{sub 4}NP-induced production of proinflammatory cytokines. Taken together, these results demonstrated that B-Fe{sub 3}O{sub 4}NPs disturb the process of autophagy in HUVECs, and eventually lead to endothelial dysfunction and inflammation.

  7. Gadd45b prevents autophagy and apoptosis against rat cerebral neuron oxygen-glucose deprivation/reperfusion injury.

    Science.gov (United States)

    He, Guoqian; Xu, Wenming; Tong, Linyan; Li, Shuaishuai; Su, Shiceng; Tan, Xiaodan; Li, Changqing

    2016-04-01

    Autophagic (type II) cell death has been suggested to play pathogenetic roles in cerebral ischemia. Growth arrest and DNA damage response 45b (Gadd45b) has been shown to protect against rat brain ischemia injury through inhibiting apoptosis. However, the relationship between Gadd45b and autophagy in cerebral ischemia/reperfusion (I/R) injury remains uncertain. The aim of this study is to investigate the effect of Gadd45b on autophagy. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) model of rat primary cortex neurons, and lentivirus interference used to silence Gadd45b expression. Cell viability and injury assay were performed using CCK-8 and LDH kit. Autophagy activation was monitored by expression of ATG5, LC3, Beclin-1, ATG7 and ATG3. Neuron apoptosis was monitored by expression of Bcl-2, Bax, cleaved caspase3, p53 and TUNEL assay. Neuron neurites were assayed by double immunofluorescent labeling with Tuj1 and LC3B. Here, we demonstrated that the expression of Gadd45b was strongly up-regulated at 24 h after 3 h OGD treatment. ShRNA-Gadd45b increased the expression of autophagy related proteins, aggravated OGD/R-induced neuron cell apoptosis and neurites injury. ShRNA-Gadd45b co-treatment with autophagy inhibitor 3-methyladenine (3-MA) or Wortmannin partly inhibited the ratio of LC3II/LC3I, and slightly ameliorated neuron cell apoptosis under OGD/R. Furthermore, shRNA-Gadd45b inhibited the p-p38 level involved in autophagy, but increased the p-JNK level involved in apoptosis. ShRNA-Gadd45b co-treatment with p38 inhibitor obviously induced autophagy. ShRNA-Gadd45b co-treatment with JNK inhibitor alleviated neuron cell apoptosis. In conclusion, our data suggested that Gadd45b inhibited autophagy and apoptosis under OGD/R. Gadd45b may be a common regulatory protein to control autophagy and apoptosis.

  8. Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

    Science.gov (United States)

    Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

    2015-07-01

    We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  9. Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

    Science.gov (United States)

    Sumis, Allison; Cook, Katherine L; Andrade, Fabia O; Hu, Rong; Kidney, Emma; Zhang, Xiyuan; Kim, Dominic; Carney, Elissa; Nguyen, Nguyen; Yu, Wei; Bouker, Kerrie B; Cruz, Idalia; Clarke, Robert; Hilakivi-Clarke, Leena

    2016-10-01

    Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight. © 2016 Society for Endocrinology.

  10. 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.

  11. Autophagy inhibition synergistically enhances anti-cancer efficacy of RAMBA, VN/12-1 in SKBR-3 cells and tumor xenografts

    Science.gov (United States)

    Godbole, Abhijit M.; Purushottamachar, Puranik; Martin, Marlena S.; Daskalakis, Constantine; Njar, Vincent C. O.

    2012-01-01

    VN/12-1 is a novel retinoic acid metabolism blocking agent (RAMBA) discovered in our laboratory. The purpose of the study was to elucidate the molecular mechanism of VN/12-1’s anticancer activity in breast cancer cell lines and in tumor xenografts. We investigated the effects of VN/12-1 on induction of autophagy andapoptosis in SKBR-3 cells. Further, we also examined the impact of pharmacological and genomic inhibition of autophagy on VN/12-1’s anti-cancer activity. Finally, the anti-tumor activity of VN/12-1 was evaluated as a single agent and in combination with autophagy inhibitor chloroquine (CHL) in an SKBR-3 mouse xenograft model. Short exposure of low dose (< 10 µM) of VN/12-1 induced endoplasmic reticulum stress (ERS), autophagy and inhibits G1-S phase transition and caused a protective response. However, higher dose of VN/12-1 initiates apoptosis in vitro. Inhibition of autophagy using either pharmacological inhibitors or RNA interference of Beclin-1 enhanced anti-cancer activity induced by VN/12-1 in SKBR-3 cells by triggering apoptosis. Importantly, VN/12-1 (5 mg/kg twice weekly) and the combination of VN/12-1 (5 mg/kg twice weekly) + chloroquine (50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (p < 0.001 vs. control) and 96.2% (p < 0.001 vs. control), respectively. Our novel findings suggest that VN/12-1 may be useful as a single agent or in combination with autophagy inhibitors for treating human breast cancers. Our data provides a strong rationale for clinical evaluation of VN/12-1 as single agent or in combination with autophagy inhibitors. PMID:22334589

  12. The post-therapeutic effect of rapamycin in mild traumatic brain-injured rats ensuing in the upregulation of autophagy and mitophagy.

    Science.gov (United States)

    Wang, Changxing; Hu, Zhiying; Zou, Yang; Xiang, Mingjun; Jiang, Yuting; Botchway, Benson O A; Huo, Xue; Du, Xiaoxue; Fang, Marong

    2017-09-01

    Mild traumatic brain injury (mTBI), common in juveniles, has been reported to be caused by sports-related concussion. Many young children may suffer from post-concussion syndrome. mTBI, in early stages of life, could play a part in neuron apoptosis and degeneration, cognitive and motor coordination impairment, as well as dementia. Our study was aimed at further investigating the post-therapeutic efficacy of rapamycin in the recuperation of mTBI while at the same time investigating the metamorphosis in both autophagy and mitophagy in mTBI. We created a weight-drop rat mTBI model with the administration of rapamycin at 4 h after every mTBI. Behavioral tests of beam walking and open field task indicated the expected improvement of cognitive and motor coordination functions. Both Western blot and immunofluorescence examinations revealed increased Beclin-1 and PINK1 in the treated rats as well as reduction of caspase-3 and cytochrome C (Cyt C). More so, the TUNEL staining evidenced curtailment of apoptotic cells following treatment with rapamycin. The upregulation of Beclin-1 and PINK1 and the downregulation of caspase-3 and Cyt C extrapolate that rapamycin plays neuroprotective as well as anti-apoptotic role via interposition of both autophagy and mitophagy. © 2017 International Federation for Cell Biology.

  13. Effect of animal mixing as a stressor on biomarkers of autophagy and oxidative stress during pig muscle maturation.

    Science.gov (United States)

    Rubio-González, A; Potes, Y; Illán-Rodríguez, D; Vega-Naredo, I; Sierra, V; Caballero, B; Fàbrega, E; Velarde, A; Dalmau, A; Oliván, M; Coto-Montes, A

    2015-07-01

    The objective of this work was to study the postmortem evolution of potential biomarkers of autophagy (Beclin 1, LC3-II/LC3-I ratio) and oxidative stress (total antioxidant activity, TAA; superoxide dismutase activity, SOD and catalase activity, CAT) in the Longissimus dorsi muscle of entire male ((Large White×Landrace)×Duroc) pigs subjected to different management treatments that may promote stress, such as mixing unfamiliar animals at the farm and/or during transport and lairage before slaughter. During the rearing period at the farm, five animals were never mixed after the initial formation of the experimental groups (unmixed group at the farm, UF), whereas 10 animals were subjected to a common routine of being mixed with unfamiliar animals (mixed group at the farm, MF). Furthermore, two different treatments were used during the transport and lairage before slaughter: 10 pigs were not mixed (unmixed group during transport and lairage, UTL), whereas five pigs were mixed with unfamiliar animals on the lorry and during lairage (mixed group during transport and lairage, MTL). These mixing treatments were then combined into three pre-slaughter treatments - namely, UF-UTL, MF-UTL and MF-MTL. The results show that MF-UTL and MF-MTL increased significantly the muscle antioxidant defense (TAA, SOD and CAT) at short postmortem times (4 and 8 h; Panimals, both at the farm and during transport and lairage, triggers postmortem muscle autophagy, which showed an earlier activation (higher expression of Beclin 1 and LC3-II/LC3-I ratio at 4 h postmortem followed by a decreasing pattern of this ratio along first 24 h postmortem) in the muscle tissues of animals from the MF-UTL and MF-MTL groups, as an adaptive strategy of the muscle cells for counteracting induced stress. From these results, we propose that monitoring the evolution of the main biomarkers of autophagy (Beclin 1, LC3-II/LC3-I ratio) and muscle antioxidant defense (TAA, SOD, CAT) in the muscle tissue within the

  14. Autophagy plays an important role in Sunitinib-mediated cell death in H9c2 cardiac muscle cells

    International Nuclear Information System (INIS)

    Zhao Yuqin; Xue Tao; Yang Xiaochun; Zhu Hong; Ding Xiaofei; Lou Liming; Lu Wei; Yang Bo; He Qiaojun

    2010-01-01

    Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity.

  15. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage.

    Science.gov (United States)

    Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

    2017-03-01

    Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1-2-3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Exogenous H2S facilitating ubiquitin aggregates clearance via autophagy attenuates type 2 diabetes-induced cardiomyopathy

    Science.gov (United States)

    Wu, Jichao; Tian, Zhiliang; Sun, Yu; Lu, Cuicui; Liu, Ning; Gao, Zhaopeng; Zhang, Linxue; Dong, Shiyun; Yang, Fan; Zhong, Xin; Xu, Changqing; Lu, Fanghao; Zhang, Weihua

    2017-01-01

    Diabetic cardiomyopathy (DCM) is a serious complication of diabetes. Hydrogen sulphide (H2S), a newly found gaseous signalling molecule, has an important role in many regulatory functions. The purpose of this study is to investigate the effects of exogenous H2S on autophagy and its possible mechanism in DCM induced by type II diabetes (T2DCM). In this study, we found that sodium hydrosulphide (NaHS) attenuated the augment in left ventricular (LV) mass and increased LV volume, decreased reactive oxygen species (ROS) production and ameliorated H2S production in the hearts of db/db mice. NaHS facilitated autophagosome content degradation, reduced the expression of P62 (a known substrate of autophagy) and increased the expression of microtubule-associated protein 1 light chain 3 II. It also increased the expression of autophagy-related protein 7 (ATG7) and Beclin1 in db/db mouse hearts. NaHS increased the expression of Kelch-like ECH-associated protein 1 (Keap-1) and reduced the ubiquitylation level in the hearts of db/db mice. 1,4-Dithiothreitol, an inhibitor of disulphide bonds, increased the ubiquitylation level of Keap-1, suppressed the expression of Keap-1 and abolished the effects of NaHS on ubiquitin aggregate clearance and ROS production in H9C2 cells treated with high glucose and palmitate. Overall, we concluded that exogenous H2S promoted ubiquitin aggregate clearance via autophagy, which might exert its antioxidative effect in db/db mouse myocardia. Moreover, exogenous H2S increased Keap-1 expression by suppressing its ubiquitylation, which might have an important role in ubiquitin aggregate clearance via autophagy. Our findings provide new insight into the mechanisms responsible for the antioxidative effects of H2S in the context of T2DCM. PMID:28796243

  17. Inhibition of acrolein-induced autophagy and apoptosis by a glycosaminoglycan from Sepia esculenta ink in mouse Leydig cells.

    Science.gov (United States)

    Gu, Yi-Peng; Yang, Xiao-Mei; Luo, Ping; Li, Yan-Qun; Tao, Ye-Xing; Duan, Zhen-Hua; Xiao, Wei; Zhang, Da-Yan; Liu, Hua-Zhong

    2017-05-01

    In our recent reports, a squid ink polysaccharide (SIP) was found having preventive activity against cyclophosphamide induced damage in mouse testis and ovary. Here we further reveal the regulative mechanism of SIP against chemical toxicity on testis. Leydig cells exposed to acrolein (ACR) underwent apoptosis at 12h and 24h. Before apoptosis, cells occurred autophagy that was confirmed by high autophagic rate and Beclin-1 protein content at 3h. PI3K/Akt and p38 MAPK signal pathways involved in the regulatory mechanisms. These outcomes of ACR were recovered completely by SIP, which was demonstrated by attenuated disruption of redox equilibrium and increased testosterone production, through suppressing ACR-caused autophagy and apoptosis regulated by PI3K/Akt and p38 MAPK signal pathways in Leydig cells. Summarily, autophagy occurred before apoptosis caused by ACR-activated p38 MAPK and PI3K/Akt pathways were blocked by SIP, resulting in survival and functional maintenance of Leydig cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

    Science.gov (United States)

    Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

    2017-10-11

    Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

  19. Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication.

    Science.gov (United States)

    Fu, Yuxuan; Xu, Wentao; Chen, Deyan; Feng, Chunhong; Zhang, Li; Wang, Xiaohui; Lv, Xiaowen; Zheng, Nan; Jin, Yu; Wu, Zhiwei

    2015-12-01

    Enterovirus 71 (EV71), the etiological agent of hand-foot-and-mouth disease, has increasingly become a public health challenge around the world. Previous studies reported that EV71 infection can induce autophagic machinery to enhance viral replication in vitro and in vivo, but did not address the underlying mechanisms. Increasing evidence suggests that autophagy, in a virus-specific manner, may function to degrade viruses or facilitate viral replication. In this study, we reported that EV71 infection of human epidermoid carcinoma (Hep2) and African green monkey kidney cells (Vero) induced autophagy, which is beneficial for viral replication. Our investigation of the mechanisms revealed that EV71 infection resulted in the reduction of cellular miR-30a, which led to the inhibition of Beclin-1, a key autophagy-promoting gene that plays important roles at the early phase of autophagosome formation. We provided further evidence that by modulating cellular miR-30a level through either overexpression or inhibition, one can inhibit or promote EV71 replication, respectively, through regulating autophagic activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Cytotoxic Autophagy in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Khushboo Sharma

    2014-06-01

    Full Text Available Autophagy is a process of cellular self-digestion, whereby the cell degrades subcellular materials in order to generate energy and metabolic precursors in order to prolong survival, classically under conditions of nutrient deprivation. Autophagy can also involve the degradation of damaged or aged organelles, and misfolded or damaged proteins to eliminate these components that might otherwise be deleterious to cellular survival. Consequently, autophagy has generally been considered a prosurvival response. Many, if not most chemotherapeutic drugs and radiation also promote autophagy, which is generally considered a cytoprotective response, in that its inhibition frequently promotes apoptotic cells death. Furthermore, it has been shown that conventional chemotherapeutic drugs and radiation alone rarely induce a form of autophagy that leads to cell death. However, there are multiple examples in the literature where newer chemotherapeutic agents, drug combinations or drugs in combination with radiation promote autophagic cell death. This review will describe autophagic cell death induced in breast tumor cells, lung cancer cells as well as glioblastoma, demonstrating that it cannot be concluded that stress induced autophagy is, of necessity, cytoprotective in function.

  1. 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.

  2. Hibiscus sabdariffa leaf polyphenolic extract induces human melanoma cell death, apoptosis, and autophagy.

    Science.gov (United States)

    Chiu, Chun-Tang; Hsuan, Shu-Wen; Lin, Hui-Hsuan; Hsu, Cheng-Chin; Chou, Fen-Pi; Chen, Jing-Hsien

    2015-03-01

    Melanoma is the least common but most fatal form of skin cancer. Previous studies have indicated that an aqueous extract of Hibiscus sabdariffa leaves possess hypoglycemic, hypolipidemic, and antioxidant effects. In this study, we want to investigate the anticancer activity of Hibiscus leaf polyphenolic (HLP) extract in melanoma cells. First, HLP was exhibited to be rich in epicatechin gallate (ECG) and other polyphenols. Apoptotic and autophagic activities of HLP and ECG were further evaluated by DAPI stain, cell-cycle analysis, and acidic vascular organelle (AVO) stain. Our results revealed that both HLP and ECG induced the caspases cleavages, Bcl-2 family proteins regulation, and Fas/FasL activation in A375 cells. In addition, we also revealed that the cells presented AVO-positive after HLP treatments. HLP could increase the expressions of autophagy-related proteins autophagy-related gene 5 (ATG5), Beclin1, and light chain 3-II (LC3-II), and induce autophagic cell death in A375 cells. These data indicated that the anticancer effect of HLP, partly contributed by ECG, in A375 cells. HLP potentially could be developed as an antimelanoma agent. © 2015 Institute of Food Technologists®

  3. Minocycline is effective in intracerebral hemorrhage by inhibition of apoptosis and autophagy.

    Science.gov (United States)

    Wu, Zehan; Zou, Xiang; Zhu, Wei; Mao, Ying; Chen, Liang; Zhao, Fan

    2016-12-15

    Intracerebral hemorrhage is the least treatable type of stroke and affects millions of people worldwide. Treatment for ICH varies from medicine to surgery, but the rate of mortality and mobility still remains high. Minocycline is a tetracycline antibiotic increasingly recognized for its neuroprotective potential. In earlier studies, we demonstrated that many secondary injuries caused by ICH could be significantly reduced by injection of minocycline in rat models. The following research investigates the role of minocycline in reducing brain injury. Twenty-four rats were administered 100μl autologous arterial blood injections into the right basal ganglia, treated with minocycline or vehicle and euthanized on the 1st, 3rd, and 7th day. Immunohistochemistry, TUNEL, and western blot analysis were performed to analyze the effects of minocycline on apoptosis and autophagy. After the injection of minocycline, TUNEL-positive cells were remarkably reduced on days 1, 3 and 7; Beclin-1, LC3BII/I, caspase-3/8 were all suppressed after treatment. The relationship between Cathepsin D and minocycline remained unknown. Our studies suggest the potential medicinal value of minocycline, through both anti-autophagy and anti-apoptosis pathways. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Local intra-articular injection of resveratrol delays cartilage degeneration in C57BL/6 mice by inducing autophagy via AMPK/mTOR pathway.

    Science.gov (United States)

    Qin, Na; Wei, Liwei; Li, Wuyin; Yang, Wei; Cai, Litao; Qian, Zhuang; Wu, Shufang

    2017-07-01

    Autophagy is an essential cellular homeostasis mechanism that was found to be compromised in aging and osteoarthritis (OA) cartilage. Previous studies showed that resveratrol can effectively regulate autophagy in other cells. The purpose of this study was to determine whether the chondroprotective effect of resveratrol was related to chondrocyte autophagy and to elucidate underlying mechanisms. OA model was induced by destabilization of the medial meniscus (DMM) in 10-week-old male mice. OA mice were treated with resveratrol with/without 3-MA for 8 weeks beginning 4 weeks after surgery. The local intra-articular injection of resveratrol delayed articular cartilage degradation in DMM-induced OA by OARSI scoring systems and Safranin O-fast green. Resveratrol treatment increased Unc-51-like kinase1, Beclin1, microtubule-associated protein light chain 3, hypoxia inducible factor-1α, phosphorylated AMPK, collagen-2A1, Aggrecan expressions, but decreased hypoxia inducible factor-2α, phosphorylated mTOR, matrix metalloproteinases13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 expressions. The effects of resveratrol were obviously blunted by 3-MA except HIF and AMPK. These findings indicate that resveratrol intra-articular injection delayed articular cartilage degeneration and promoted chondrocyte autophagy in an experimental model of surgical DMM-induced OA, in part via balancing HIF-1α and HIF-2α expressions and thereby regulating AMPK/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  5. Autophagy in human embryonic stem cells

    NARCIS (Netherlands)

    Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

    2011-01-01

    Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of

  6. Enhanced myometrial autophagy in postpartum uterine involution

    Directory of Open Access Journals (Sweden)

    Keng-Fu Hsu

    2014-09-01

    Conclusion: Autophagy of myocytes may play an important role in uterine involution. These results have implications for our understanding of myometrial functional adaptations during pregnancy and the physiological role of autophagy in the uterine remodeling events in the postpartum period.

  7. Osteopontin Promotes Cell Migration and Invasion, and Inhibits Apoptosis and Autophagy in Colorectal Cancer by activating the p38 MAPK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ren-hong Huang

    2017-04-01

    Full Text Available Background: Osteopontin (OPN is highly expressed in colorectal cancer (CRC and is associated with disease progression in vivo. High levels of OPN have been demonstrated to predict low survival rates in CRC. Autophagy is a process of self-digestion, which is thought to play a significant role in carcinogenesis. However, the mechanisms of OPN's effects on CRC cell autophagy have not been elucidated. Therefore, we aimed to investigate possible mechanisms of OPN's effects on CRC autophagy. Methods: HCT116 cell proliferation, apoptosis, and migration and invasion ability were identified by cell counting k¡t-8 assay, flow cytometry, wound healing assay, and transwell chamber invasion assay, respectively. The ratios of proteins LC3-II/LC3-I, P62, and Atg7 were analyzed by Western-blot. Expressions of Beclin-1, Atg4b, Bnip3, and Vps34, both in transcriptional and translational levels, were analyzed and compared by RT-PCR and Western blot. Immunofluorescence and co-focusing experiments were used to investigate the formation of autophagosomes. Results: The results showed that OPN can promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis. It was also demonstrated that OPN could inhibit cell autophagy. Further experiments revealed that the inhibitory effect of OPN on autophagy could be reversed by blocking the p38 MAPK pathway in HCT116 cells. Conclusion: OPN is involved in HCT116 cell progression and is capable of inhibiting cell autophagy possibly by activating the p38 MAPK signaling pathway, implying that OPN could be a potential novel molecular therapeutic biomarker in patients with CRC.

  8. Transportin-1-dependent YB-1 nuclear import

    International Nuclear Information System (INIS)

    Mordovkina, Daria A.; Kim, Ekaterina R.; Buldakov, Ilya A.; Sorokin, Alexey V.; Eliseeva, Irina A.; Lyabin, Dmitry N.; Ovchinnikov, Lev P.

    2016-01-01

    The DNA/RNA-binding protein YB-1 (Y-box binding protein 1) performs multiple functions both in the cytoplasm and the nucleus of the cell. Generally localized to the cytoplasm, under certain conditions YB-1 is translocated to the nucleus. Here we report for the first time a transport factor that mediates YB-1 nuclear import – transportin-1. The YB-1/transportin-1 complex can be isolated from HeLa cell extract. Nuclear import of YB-1 and its truncated form YB-1 (1-219) in in vitro transport assay was diminished in the presence of a competitor substrate and ceased in the presence of transportin-1 inhibitor M9M. Inhibitors of importin β1 had no effect on YB-1 transport. Furthermore, transport of YB-1 (P201A/Y202A) and YB-1 (1–219) (P201A/Y202A) bearing inactivating mutations in the transportin-1-dependent nuclear localization signal was practically abolished. Together, these results indicate that transportin-1 mediates YB-1 nuclear translocation. - Highlights: • Transportin-1 mediates YB-1 nuclear import. • YB-1 nuclear translocation is diminished in the presence of transportin-1 inhibitors. • Mutations in the PY motif of YB-1 NLS prevent its translocation to the nucleus.

  9. Association of FKBP51 with priming of autophagy pathways and mediation of antidepressant treatment response: evidence in cells, mice, and humans.

    Directory of Open Access Journals (Sweden)

    Nils C Gassen

    2014-11-01

    Full Text Available FK506 binding protein 51 (FKBP51 is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways. Importantly, the clinical antidepressant response of patients with depression (n = 51 could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r

  10. Mesenchymal stem cells enhance autophagy and increase β-amyloid clearance in Alzheimer disease models

    Science.gov (United States)

    Shin, Jin Young; Park, Hyun Jung; Kim, Ha Na; Oh, Se Hee; Bae, Jae-Sung; Ha, Hee-Jin; Lee, Phil Hyu

    2014-01-01

    Current evidence suggests a central role for autophagy in Alzheimer disease (AD), and dysfunction in the autophagic system may lead to amyloid-β (Aβ) accumulation. Using in vitro and in vivo AD models, the present study investigated whether mesenchymal stem cells (MSCs) could enhance autophagy and thus exert a neuroprotective effect through modulation of Aβ clearance In Aβ-treated neuronal cells, MSCs increased cellular viability and enhanced LC3-II expression compared with cells treated with Aβ only. Immunofluorescence revealed that MSC coculture in Aβ-treated neuronal cells increased the number of LC3-II-positive autophagosomes that were colocalized with a lysosomal marker. Ultrastructural analysis revealed that most autophagic vacuoles (AVs) in Aβ-treated cells were not fused with lysosomes, whereas a large portion of autophagosomes were conjoined with lysosomes in MSCs cocultured with Aβ-treated neuronal cells. Furthermore, MSC coculture markedly increased Aβ immunoreactivity colocalized within lysosomes and decreased intracellular Aβ levels compared with Aβ-treated cells. In Aβ-treated animals, MSC administration significantly increased autophagosome induction, final maturation of late AVs, and fusion with lysosomes. Moreover, MSC administration significantly reduced the level of Aβ in the hippocampus, which was elevated in Aβ-treated mice, concomitant with increased survival of hippocampal neurons. Finally, MSC coculture upregulated BECN1/Beclin 1 expression in AD models. These results suggest that MSCs significantly enhance autolysosome formation and clearance of Aβ in AD models, which may lead to increased neuronal survival against Aβ toxicity. Modulation of the autophagy pathway to repair the damaged AD brain using MSCs would have a significant impact on future strategies for AD treatment. PMID:24149893

  11. Effects of chronic sugar consumption on lipid accumulation and autophagy in the skeletal muscle.

    Science.gov (United States)

    De Stefanis, Daniela; Mastrocola, Raffaella; Nigro, Debora; Costelli, Paola; Aragno, Manuela

    2017-02-01

    In recent years, the increasing consumption of soft drinks containing high-fructose corn syrup or sucrose has caused a rise in fructose intake, which has been related to the epidemic of metabolic diseases. As fructose and glucose intake varies in parallel, it is still unclear what the effects of the increased consumption of the two single sugars are. In the present study, the impact of chronic consumption of glucose or fructose on skeletal muscle of healthy mice was investigated. C57BL/6J male mice received water (C), 15 % fructose (ChF) or 15 % glucose (ChG) to drink for up to 7 months. Lipid metabolism and markers of inflammation and autophagy were assessed in gastrocnemius muscle. Increased body weight and gastrocnemius muscle mass, as well as circulating glucose, insulin, and lipid plasma levels were observed in sugar-drinking mice. Although triglycerides increased in the gastrocnemius muscle of both ChF and ChG mice (+32 and +26 %, vs C, respectively), intramyocellular lipids accumulated to a significantly greater extent in ChF than in ChG animals (ChF +10 % vs ChG). Such perturbations were associated with increased muscle interleukin-6 levels (threefold of C) and with the activation of autophagy, as demonstrated by the overexpression of LC3B-II (ChF, threefold and ChG, twofold of C) and beclin-1 (ChF, sevenfold and ChG, tenfold of C). The present results suggest that intramyocellular lipids and the pro-inflammatory signaling could contribute to the onset of insulin resistance and lead to the induction of autophagy, which could be an adaptive response to lipotoxicity.

  12. miR-26a suppresses autophagy in swine Sertoli cells by targeting ULK2.

    Science.gov (United States)

    Ran, M; Li, Z; Cao, R; Weng, B; Peng, F; He, C; Chen, B

    2018-05-14

    A large number of microRNAs (miRNAs) have been detected from porcine testicular tissues thanks to the development of high-throughput sequencing technology. However, the regulatory roles of most identified miRNAs in swine testicular development or spermatogenesis are poorly understood. In our previous study, ULK2 (uncoordinated-51-like kinase 2) was predicted as a target gene of miR-26a. In this study, we aimed to investigate the role of miR-26a in swine Sertoli cell autophagy. The relative expression of miR-26a and ULK2 levels has a significant negative correlation (R 2  = .5964, p ≤ .01) in nine developmental stages of swine testicular tissue. Dual-luciferase reporter assay results show that miR-26a directly targets the 3'UTR of the ULK2 gene (position 618-624). In addition, both the mRNA and protein expression of ULK2 were downregulated by miR-26a in swine Sertoli cells. These results indicate that miR-26a targets the ULK2 gene and downregulates its expression in swine Sertoli cells. Based on the expression of marker genes (LC3, p62 and Beclin-1), overexpression of miR-26a or knock-down of ULK2 inhibits swine Sertoli cell autophagy. Taken together, these findings demonstrate that miR-26a suppresses autophagy in swine Sertoli cells by targeting ULK2. © 2018 Blackwell Verlag GmbH.

  13. Curcumin Inhibits Apoptosis of Chondrocytes through Activation ERK1/2 Signaling Pathways Induced Autophagy

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    Xiaodong Li

    2017-04-01

    Full Text Available Osteoarthritis (OA is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective in treating pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe, and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Curcumin, the principal curcuminoid and the most active component in turmeric, is a biologically active phytochemical. Evidence from several recent in vitro studies suggests that curcumin may exert a chondroprotective effect through actions such as anti-inflammatory, anti-oxidative stress, and anti-catabolic activity that are critical for mitigating OA disease pathogenesis and symptoms. In the present study, we investigated the protective mechanisms of curcumin on interleukin 1β (IL-1β-stimulated primary chondrocytes in vitro. The treatment of interleukin (IL-1β significantly reduces the cell viability of chondrocytes in dose and time dependent manners. Co-treatment of curcumin with IL-1β significantly decreased the growth inhibition. We observed that curcumin inhibited IL-1β-induced apoptosis and caspase-3 activation in chondrocytes. Curcumin can increase the expression of phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2, autophagy marker light chain 3 (LC3-II, and Beclin-1 in chondrocytes. The expression of autophagy markers could be decreased when the chondrocytes were incubated with ERK1/2 inhibitor U0126. Our results suggest that curcumin suppresses apoptosis and inflammatory signaling through its actions on the ERK1/2-induced autophagy in chondrocytes. We propose that curcumin should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

  14. Exploring autophagy with Gene Ontology

    Science.gov (United States)

    2018-01-01

    ABSTRACT Autophagy is a fundamental cellular process that is well conserved among eukaryotes. It is one of the strategies that cells use to catabolize substances in a controlled way. Autophagy is used for recycling cellular components, responding to cellular stresses and ridding cells of foreign material. Perturbations in autophagy have been implicated in a number of pathological conditions such as neurodegeneration, cardiac disease and cancer. The growing knowledge about autophagic mechanisms needs to be collected in a computable and shareable format to allow its use in data representation and interpretation. The Gene Ontology (GO) is a freely available resource that describes how and where gene products function in biological systems. It consists of 3 interrelated structured vocabularies that outline what gene products do at the biochemical level, where they act in a cell and the overall biological objectives to which their actions contribute. It also consists of ‘annotations’ that associate gene products with the terms. Here we describe how we represent autophagy in GO, how we create and define terms relevant to autophagy researchers and how we interrelate those terms to generate a coherent view of the process, therefore allowing an interoperable description of its biological aspects. We also describe how annotation of gene products with GO terms improves data analysis and interpretation, hence bringing a significant benefit to this field of study. PMID:29455577

  15. Intraoperative hemidiaphragm electrical stimulation reduces oxidative stress and upregulates autophagy in surgery patients undergoing mechanical ventilation: exploratory study

    Directory of Open Access Journals (Sweden)

    Robert T. Mankowski

    2016-10-01

    Full Text Available Abstract Background Mechanical ventilation (MV during a cardio-thoracic surgery contributes to diaphragm muscle dysfunction that impairs weaning and can lead to the ventilator- induced diaphragm dysfunction. Especially, it is critical in older adults who have lower muscle reparative capacity following MV. Reports have shown that the intraoperative intermittent hemidiaphragm electrical stimulation can maintain and/or improve post-surgery diaphragm function. In particular, from a molecular point of view, intermittent electrical stimulation (ES may reduce oxidative stress and increase regulatory autophagy levels, and therefore improve diaphragm function in animal studies. We have recently shown in humans that intraoperative ES attenuates mitochondrial dysfunction and force decline in single diaphragm muscle fibers. The aim of this study was to investigate an effect of ES on oxidative stress, antioxidant status and autophagy biomarker levels in the human diaphragm during surgery. Methods One phrenic nerve was simulated with an external cardiac pacer in operated older subjects (62.4 ± 12.9 years (n = 8 during the surgery. The patients received 30 pulses per min every 30 min. The muscle biopsy was collected from both hemidiaphragms and frozen for further analyses. 4-hydroxynonenal (4-HNE, an oxidative stress marker, and autophagy marker levels (Beclin-1 and the ratio of microtubule-associated protein light chain 3, I and II-LC3 II/I protein concentrations were detected by the Western Blot technique. Antioxidant enzymatic activity copper-zinc (CuZnSOD and manganese (MnSOD superoxide dismutase were analyzed. Results Levels of lipid peroxidation (4-HNE were significantly lower in the stimulated side (p  0.05. Additionally, the protein concentrations of Beclin-1 and the LC3 II/I ratio were higher in the stimulated side (p < 0.05. Conclusion These results suggest that the intraoperative electrical stimulation decreases oxidative stress levels

  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. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Role of D-Limonene in Autophagy Induced by Bergamot Essential Oil in SH-SY5Y Neuroblastoma Cells

    Science.gov (United States)

    Russo, Rossella; Cassiano, Maria Gilda Valentina; Ciociaro, Antonella; Adornetto, Annagrazia; Varano, Giuseppe Pasquale; Chiappini, Carlotta; Berliocchi, Laura; Tassorelli, Cristina; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2014-01-01

    Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005–0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70S6K (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125–750 µM) and linalyl acetate (62.5–375 µM), were individually tested at concentrations comparable to those found in 0.005–0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by d-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by d

  18. Ordered bulk degradation via autophagy

    DEFF Research Database (Denmark)

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

    2008-01-01

    During amino acid starvation, cells undergo macroautophagy which is regarded as an unspecific bulk degradation process. Lately, more and more organelle-specific autophagy subtypes such as reticulophagy, mitophagy and ribophagy have been described and it could be shown, depending on the experimental...... 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...

  19. Autophagy, lipophagy and lysosomal lipid storage disorders.

    Science.gov (United States)

    Ward, Carl; Martinez-Lopez, Nuria; Otten, Elsje G; Carroll, Bernadette; Maetzel, Dorothea; Singh, Rajat; Sarkar, Sovan; Korolchuk, Viktor I

    2016-04-01

    Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Modulation of pathogen recognition by autophagy

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    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.

  1. Nobel Prize Honors Autophagy Discovery.

    Science.gov (United States)

    2016-12-01

    Japanese cell biologist Yoshinori Ohsumi, PhD, was awarded this year's Nobel Prize in Physiology or Medicine for his discovery of autophagy. His groundbreaking studies in yeast cells illuminated how cells break down and recycle damaged material, a process that is critical to the survival of both normal cells and some cancer cells. ©2016 American Association for Cancer Research.

  2. Human Papilloma Virus and Autophagy

    Directory of Open Access Journals (Sweden)

    Domenico Mattoscio

    2018-06-01

    Full Text Available Human papilloma viruses (HPVs are a group of double-stranded DNA viruses known to be the primary cause of cervical cancer. In addition, evidence has now established their role in non-melanoma skin cancers, head and neck cancer (HNC, and the development of other anogenital malignancies. The prevalence of HPV-related HNC, in particular oropharyngeal cancers, is rapidly increasing, foreseeing that HPV-positive oropharyngeal cancers will outnumber uterine cervical cancers in the next 15–20 years. Therefore, despite the successful advent of vaccines originally licensed for cervical cancer prevention, HPV burden is still very high, and a better understanding of HPV biology is urgently needed. Autophagy is the physiological cellular route that accounts for removal, degradation, and recycling of damaged organelles, proteins, and lipids in lysosomal vacuoles. In addition to this scavenger function, autophagy plays a fundamental role during viral infections and cancers and is, therefore, frequently exploited by viruses to their own benefit. Recently, a link between HPV and autophagy has clearly emerged, leading to the conceivable development of novel anti-viral strategies aimed at restraining HPV infectivity. Here, recent findings on how oncogenic HPV16 usurp autophagy are described, highlighting similarities and differences with mechanisms adopted by other oncoviruses.

  3. Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition

    Science.gov (United States)

    Park, Dohyun; Jeong, Heeyoon; Lee, Mi Nam; Koh, Ara; Kwon, Ohman; Yang, Yong Ryoul; Noh, Jungeun; Suh, Pann-Ghill; Park, Hwangseo; Ryu, Sung Ho

    2016-01-01

    Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction. PMID:26902888

  4. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-11-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

  5. 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.

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    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.

  6. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Moussa, Mayssam, E-mail: Moussa-mayssam@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Lajeunesse, Daniel, E-mail: daniel.lajeunesse@umontreal.ca [Research Centre in Osteoarthritis, Research Centre in Monteral University (Canada); Hilal, George, E-mail: George2266@gmail.com [Cancer and metabolism Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); El Atat, Oula, E-mail: oulaatat@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Haykal, Gaby, E-mail: Gaby.haykal@hdf.usj.edu.lb [Hotel Dieu de France, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Serhal, Rim, E-mail: rim.basbous@gmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Chalhoub, Antonio, E-mail: Mava.o@hotmail.com [Carantina Hospital, Beirut (Lebanon); Khalil, Charbel, E-mail: charbelk3@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Alaaeddine, Nada, E-mail: Nada.aladdin@gmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon)

    2017-03-01

    Objectives: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Methods: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1–2–3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. Results: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. Conclusion: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. - Highlights: • Platelet Rich Plasma is suggested as a new treatment for osteoarthritis. • The proposed therapeutic effect is

  7. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage

    International Nuclear Information System (INIS)

    Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

    2017-01-01

    Objectives: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Methods: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1–2–3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. Results: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. Conclusion: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. - Highlights: • Platelet Rich Plasma is suggested as a new treatment for osteoarthritis. • The proposed therapeutic effect is

  8. Kaempferol alleviates ox-LDL-induced apoptosis by up-regulation of autophagy via inhibiting PI3K/Akt/mTOR pathway in human endothelial cells.

    Science.gov (United States)

    Che, Jianbo; Liang, Bing; Zhang, Yuan; Wang, Yi; Tang, Jianyu; Shi, Gongning

    Oxidized low-density lipoprotein (ox-LDL) has been reported to induce apoptosis of endothelial cells (ECs) and contribute to the progression of atherosclerosis. Kaempferol has been shown to possess antiatherosclerotic effect. The aim of the present study was to evaluate the effect of kaempferol on ox-LDL-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and its possible molecular basis. The results showed that kaempferol alleviated ox-LDL-induced apoptosis. Kaempferol increased the ratio of LC3-II/I and beclin-1 level in ox-LDL-induced HUVECs. Moreover, the expression of p-Akt and p-mTOR was down-regulated after treatment with kaempferol in ox-LDL-treated HUVECs, which is similar to the effect of PI3K inhibitor (LY294002) or mTOR inhibitor [rapamycin (RAP)]. Besides, autophagy induced by kaempferol was enhanced by LY294002 or RAP, while kaempferol-induced autophagy was attenuated with insulin treatment, the activator of PI3K/Akt/mTOR pathway. Furthermore, insulin also abated the effect of kaempferol on cell viability and apoptosis in ox-LDL-induced HUVECs. The results indicated that kaempferol alleviated ox-LDL-induced cell apoptosis by up-regulation of autophagy via inhibiting PI3K/Akt/mTOR pathway in human ECs. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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.

  10. Autophagy is essential for hearing in mice.

    Science.gov (United States)

    Fujimoto, Chisato; Iwasaki, Shinichi; Urata, Shinji; Morishita, Hideaki; Sakamaki, Yuriko; Fujioka, Masato; Kondo, Kenji; Mizushima, Noboru; Yamasoba, Tatsuya

    2017-05-11

    Hearing loss is the most frequent sensory disorder in humans. Auditory hair cells (HCs) are postmitotic at late-embryonic differentiation and postnatal stages, and their damage is the major cause of hearing loss. There is no measurable HC regeneration in the mammalian cochlea, and the maintenance of cell function is crucial for preservation of hearing. Here we generated mice deficient in autophagy-related 5 (Atg5), a gene essential for autophagy, in the HCs to investigate the effect of basal autophagy on hearing acuity. Deletion of Atg5 resulted in HC degeneration and profound congenital hearing loss. In autophagy-deficient HCs, polyubiquitinated proteins and p62/SQSTM1, an autophagy substrate, accumulated as inclusion bodies during the first postnatal week, and these aggregates increased in number. These findings revealed that basal autophagy has an important role in maintenance of HC morphology and hearing acuity.

  11. Osteoporosis and autophagy: What is the relationship?

    Directory of Open Access Journals (Sweden)

    Rinaldo Florencio-Silva

    Full Text Available Summary Autophagy is a survival pathway wherein non-functional proteins and organelles are degraded in lysosomes for recycling and energy production. Therefore, autophagy is fundamental for the maintenance of cell viability, acting as a quality control process that prevents the accumulation of unnecessary structures and oxidative stress. Increasing evidence has shown that autophagy dysfunction is related to several pathologies including neurodegenerative diseases and cancer. Moreover, recent studies have shown that autophagy plays an important role for the maintenance of bone homeostasis. For instance, in vitro and animal and human studies indicate that autophagy dysfunction in bone cells is associated with the onset of bone diseases such as osteoporosis. This review had the purpose of discussing the issue to confirm whether a relationship between autophagy dysfunction and osteoporosis exits.

  12. Autophagy in the control of food intake

    OpenAIRE

    Singh, Rajat

    2012-01-01

    The cellular nutrient sensing apparatus detects nutritional depletion and transmits this information to downstream effectors that generate energy from alternate sources. Autophagy is a crucial catabolic pathway that turns over redundant cytoplasmic components in lysosomes to provide energy to the starved cell. Recent studies have described a role for hypothalamic autophagy in the control of food intake and energy balance. Activated autophagy in hypothalamic neurons during starvation mobilized...

  13. 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.

  14. 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. Copyright © 2016 the American Physiological Society.

  15. Emerging connections between RNA and autophagy

    DEFF Research Database (Denmark)

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

    2017-01-01

    in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge......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 the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease....

  16. 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.

  17. Approaches for Studying Autophagy in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Yanfang Chen

    2017-08-01

    Full Text Available Macroautophagy (hereafter referred to as autophagy is an intracellular degradative process, well conserved among eukaryotes. By engulfing cytoplasmic constituents into the autophagosome for degradation, this process is involved in the maintenance of cellular homeostasis. Autophagy induction triggers the formation of a cup-shaped double membrane structure, the phagophore, which progressively elongates and encloses materials to be removed. This double membrane vesicle, which is called an autophagosome, fuses with lysosome and forms the autolysosome. The inner membrane of the autophagosome, along with engulfed compounds, are degraded by lysosomal enzymes, which enables the recycling of carbohydrates, amino acids, nucleotides, and lipids. In response to various factors, autophagy can be induced for non-selective degradation of bulk cytoplasm. Autophagy is also able to selectively target cargoes and organelles such as mitochondria or peroxisome, functioning as a quality control system. The modification of autophagy flux is involved in developmental processes such as resistance to stress conditions, aging, cell death, and multiple pathologies. So, the use of animal models is essential for understanding these processes in the context of different cell types throughout the entire lifespan. For almost 15 years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. This review presents a rapid overview of physiological processes involving autophagy in Caenorhabditis elegans, the different assays used to monitor autophagy, their drawbacks, and specific tools for the analyses of selective autophagy.

  18. 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.

  19. 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

  20. Activating Nrf-2 signaling depresses unilateral ureteral obstruction-evoked mitochondrial stress-related autophagy, apoptosis and pyroptosis in kidney.

    Directory of Open Access Journals (Sweden)

    Shue Dong Chung

    Full Text Available Exacerbated oxidative stress and inflammation may induce three types of programmed cell death, autophagy, apoptosis and pyroptosis in unilateral ureteral obstruction (UUO kidney. Sulforaphane activating NF-E2-related nuclear factor erythroid-2 (Nrf-2 signaling may ameliorate UUO-induced renal damage. UUO was induced in the left kidney of female Wistar rats. The level of renal blood flow, cortical and medullary oxygen tension and reactive oxygen species (ROS was evaluated. Fibrosis, ED-1 (macrophage/monocyte infiltration, oxidative stress, autophagy, apoptosis and pyroptosis were evaluated by immunohistochemistry and Western blot in UUO kidneys. Effects of sulforaphane, an Nrf-2 activator, on Nrf-2- and mitochondrial stress-related proteins and renal injury were examined. UUO decreased renal blood flow and oxygen tension and increased renal ROS, 3-nitrotyrosine stain, ED-1 infiltration and fibrosis. Enhanced renal tubular Beclin-1 expression started at 4 h UUO and further enhanced at 3d UUO, whereas increased Atg-5-Atg12 and LC3-II expression were found at 3d UUO. Increased renal Bax/Bcl-2 ratio, caspase 3 and PARP fragments, apoptosis formation associated with increased caspase 1 and IL-1β expression for pyroptosis formation were started from 3d UUO. UUO reduced nuclear Nrf-2 translocation, increased cytosolic and inhibitory Nrf-2 expression, increased cytosolic Bax translocation to mitochondrial and enhanced mitochondrial Cytochrome c release into cytosol of the UUO kidneys. Sulforaphane significantly increased nuclear Nrf-2 translocation and decreased mitochondrial Bax translocation and Cytochrome c release into cytosol resulting in decreased renal injury. In conclusion, sulforaphane via activating Nrf-2 signaling preserved mitochondrial function and suppressed UUO-induced renal oxidative stress, inflammation, fibrosis, autophagy, apoptosis and pyroptosis.

  1. The endoplasmic reticulum stress-autophagy pathway is involved in apelin-13-induced cardiomyocyte hypertrophy in vitro

    Institute of Scientific and Technical Information of China (English)

    Feng XIE; Di WU; Shi-fang HUANG; Jian-gang CAO; He-ning LI; Lu HE; Mei-qing LIU; Lan-fang LI; Lin-xi CHEN

    2017-01-01

    Apelin is the endogenous ligand for the G protein-coupled receptor APJ,and plays important roles in the cardiovascular system.Our previous studies showed that apelin-13 promotes the hypertrophy of H9c2 rat cardiomyocytes through the PI3K-autophagy pathway.The aim of this study was to explore what roles ER stress and autophagy played in apelin-13-induced hypertrophy of cardiomyocytes in vitro.Treatment of H9c2 cells with apelin-13 (0.001-2 μJmol/L) dose-dependently increased the production of ROS and the expression levels of NADPH oxidase 4 (NOX4).Knockdown of Nox4 with siRNAs effectively prevented the reduction of GSH/GSSG ratio in apelin-13-treated cells.Furthermore,apelin-13 treatment dose-dependently increased the expression of Bip and CHOP,two ER stress markers,in the cells.Knockdown of APJ or Nox4 with the corresponding siRNAs,or application of NADPH inhibitor DPI blocked apelin-13-induced increases in Bip and CHOP expression.Moreover,apelin-13 treatment increased the formation of autophagosome and ER fragments and the LC3 puncta in the ER of the cells.Knockdown of APJ,Nox4,Bip or CHOP with the corresponding siRNAs,or application of DPI or salubrinal attenuated apelin-13-induced overexpression of LC3-Ⅱ/Ⅰ and beclin 1.Finally,knockdown of Nox4,Bip or CHOP with the corresponding siRNAs,or application of salubrinal significantly suppressed apelin-13-induced increases in the cell diameter,volume and protein contents.Our results demonstrate that ER stress-autophagy is involved in apelin-13-induced H9c2 cell hypertrophy.

  2. Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy.

    Directory of Open Access Journals (Sweden)

    Grant R Campbell

    Full Text Available Low vitamin D levels in human immunodeficiency virus type-1 (HIV infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3, the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A₁, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.

  3. 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.

  4. 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.

  5. 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.

  6. Autophagy: A Sweet Process in Diabetes

    NARCIS (Netherlands)

    Meijer, Alfred J.; Codogno, Patrice

    2008-01-01

    Autophagy is inhibited by the insulin-amino acid-mTOR signaling pathway. Two papers in this issue of Cell Metabolism (Ebato et al., 2008; Jung et al., 2008) provide evidence that basal autophagy is necessary to maintain the architecture and function of pancreatic beta cells and that its induction in

  7. Autophagy Proteins in Phagocyte Endocytosis and Exocytosis

    Directory of Open Access Journals (Sweden)

    Christian Münz

    2017-09-01

    Full Text Available Autophagy was initially described as a catabolic pathway that recycles nutrients of cytoplasmic constituents after lysosomal degradation during starvation. Since the immune system monitors products of lysosomal degradation via major histocompatibility complex (MHC class II restricted antigen presentation, autophagy was found to process intracellular antigens for display on MHC class II molecules. In recent years, however, it has become apparent that the molecular machinery of autophagy serves phagocytes in many more membrane trafficking pathways, thereby regulating immunity to infectious disease agents. In this minireview, we will summarize the recent evidence that autophagy proteins regulate phagocyte endocytosis and exocytosis for myeloid cell activation, pathogen replication, and MHC class I and II restricted antigen presentation. Selective stimulation and inhibition of the respective functional modules of the autophagy machinery might constitute valid therapeutic options in the discussed disease settings.

  8. PICALM modulates autophagy activity and tau accumulation

    Science.gov (United States)

    Moreau, Kevin; Fleming, Angeleen; Imarisio, Sara; Lopez Ramirez, Ana; Mercer, Jacob L.; Jimenez-Sanchez, Maria; Bento, Carla F.; Puri, Claudia; Zavodszky, Eszter; Siddiqi, Farah; Lavau, Catherine P.; Betton, Maureen; O’Kane, Cahir J.; Wechsler, Daniel S.; Rubinsztein, David C.

    2014-01-01

    Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover. PMID:25241929

  9. Autophagy in endometriosis: Friend or foe?

    Science.gov (United States)

    Zhan, Lei; Li, Jun; Wei, Bing

    2018-01-01

    Endometriosis is a chronic, estrogen-dependent disease and characterized by the implantation of endometrial glands and stroma deep and haphazardly into the outside the uterine cavity. It affects an estimated 10% of the female population of reproductive age and results in obvious reduction in health-related quality of life. Unfortunately, there is no a consistent theory for the etiology of endometriosis. Furthermore, the endometriosis is hard to diagnose in early stage and the treatment methods are limited. Importantly, emerging evidence has investigated that there is a close relationship between endometriosis and autophagy. However, autophagy is a friend or foe in endometriosis is puzzling, the precise mechanism underlying autophagy in endometriosis has not been fully elucidated yet. Here, we provide an integrated view on the acquired findings of the connections between endometriosis and autophagy. We also discuss which may contribute to the abnormal level of autophagy in endometriosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. WNK1 is an unexpected autophagy inhibitor

    Science.gov (United States)

    Gallolu Kankanamalage, Sachith; Lee, A-Young; Wichaidit, Chonlarat; Lorente-Rodriguez, Andres; Shah, Akansha M.; Stippec, Steve; Whitehurst, Angelique W.; Cobb, Melanie H.

    2017-01-01

    ABSTRACT Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions. PMID:28282258

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

    OpenAIRE

    Mostowy, Serge

    2012-01-01

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

  12. The induction of apoptosis and autophagy in human hepatoma SMMC-7721 cells by combined treatment with vitamin C and polysaccharides extracted from Grifola frondosa.

    Science.gov (United States)

    Zhao, Fei; Zhao, Jin; Song, Lei; Zhang, Ya-Qing; Guo, Zhong; Yang, Ke-Hu

    2017-11-01

    Polysaccharides extracted from the mushroom Grifola frondosa (GFP) are a potential anticancer agent. The objective of this study was to investigate the effect of GFP and vitamin C (VC) alone and in combination on the viability of human hepatocarcinoma SMMC-7721 and HepG2 cells. Studies designed to detect cell apoptosis and autophagy were also conducted to investigate the mechanism. Results from the cell viability assay indicated that a combination of GFP (0.2 or 0.25 mg/mL) and VC (0.3 mmol/L) (GFP/VC) led to 52.73 and 53.93% reduction in cell viability of SMMC-7721 and HepG2 cells separately after 24 h. Flow cytometric analysis indicated that GFP/VC treatment induced cell cycle arrest at the G2/M phase, and apoptosis occurred in approximately 43.62 and 42.46% of the SMMC-7721 and HepG2 cells separately. Moreover, results of Hoechst33258 and monodansylcadaverine staining, and transmission electron microscopy, showed that GFP/VC induced apoptosis and autophagy in SMMC-7721 and HepG2 cells. Western blot analysis showed changes in the expression of apoptosis-related proteins [upregulation of BAX and caspase-3, downregulation of Bcl-2, and activation of poly-(ADP-ribose)-polymerase] and autophagy protein markers (upregulation of beclin-1 and microtubule-associated protein 1A/1B light chain-3). We also demonstrated that the expression of both Akt and p-Akt was enhanced, suggesting the PI3K/Akt/mTOR pathway might not be involved in this process. Our study shows that the combined application of GFP and VC induced cell apoptosis and autophagy in vitro, and might have antitumor activity in vivo.

  13. Exposure to 16O-particle radiation causes aging-like decrements in rats through increased oxidative stress, inflammation and loss of autophagy.

    Science.gov (United States)

    Poulose, Shibu M; Bielinski, Donna F; Carrihill-Knoll, Kirsty; Rabin, Bernard M; Shukitt-Hale, Barbara

    2011-12-01

    Exposing young rats to particles of high energy and charge (HZE particles), a ground-based model for exposure to cosmic rays, enhances indices of oxidative stress and inflammation, disrupts the functioning of neuronal communication, and alters cognitive behaviors. Even though exposure to HZE particles occurs at low fluence rates, the cumulative effects of long-term exposure result in molecular changes similar to those seen in aged animals. In the present study, we assessed markers of autophagy, a dynamic process for intracellular degradation and recycling of toxic proteins and organelles, as well as stress and inflammatory responses, in the brains of Sprague-Dawley rats irradiated at 2 months of age with 5 and 50 cGy and 1 Gy of ionizing oxygen particles ((16)O) (1000 MeV/n). Compared to nonirradiated controls, exposure to (16)O particles significantly inhibited autophagy function in the hippocampus as measured by accumulation of ubiquitin inclusion bodies such as P62/SQSTM1, autophagosome marker microtubule-associated protein 1 beta light chain 3 (MAP1B-LC3), beclin1 and proteins such as mammalian target of rapamycin (mTOR). The molecular changes measured at short (36 h) and long (75 days) intervals after (16)O-particle exposure indicate that the loss of autophagy function occurred shortly after exposure but was recovered via inhibition of mTOR. However, HZE-particle radiation caused significant sustained loss of protein kinase C alpha (PKC-α), a key G protein modulator involved in neuronal survival and functions of neuronal trophic factors. Exposure to (16)O particles also caused substantial increases in the levels of nuclear factor kappa B (NF-κB) and glial fibrillary acidic protein (GFAP), indicating glial cell activation 75 days after exposure. This is the first report to show the molecular effects of (16)O-particle radiation on oxidative stress, inflammation and loss of autophagy in the brain of young rats.

  14. High Endogenous Accumulation of ω-3 Polyunsaturated Fatty Acids Protect against Ischemia-Reperfusion Renal Injury through AMPK-Mediated Autophagy in Fat-1 Mice

    Directory of Open Access Journals (Sweden)

    Do Hyeong Gwon

    2017-09-01

    Full Text Available Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI. Fat-1 transgenic mice produce ω3-Polyunsaturated fatty acids (ω3-PUFAs from ω6-Polyunsaturated fatty acids (ω6-PUFAs without a dietary ω3-PUFAs supplement, leading to a high accumulation of omega-3 in various tissues. ω3-PUFAs show protective effects against various renal injuries and it has recently been reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI and evaluated its associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt were divided into four groups: wt sham (n = 10, fat-1 sham (n = 10, wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15, and fat-1 IRI (n = 15. Kidneys and blood were harvested 24 h after IRI and renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3-II, Beclin-1, and Atg7; lower amounts of p62; and, higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more adenosine monophosphate-activated protein kinase (AMPK activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR. Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.

  15. High Endogenous Accumulation of ω-3 Polyunsaturated Fatty Acids Protect against Ischemia-Reperfusion Renal Injury through AMPK-Mediated Autophagy in Fat-1 Mice.

    Science.gov (United States)

    Gwon, Do Hyeong; Hwang, Tae Woong; Ro, Ju-Ye; Kang, Yoon-Joong; Jeong, Jin Young; Kim, Do-Kyung; Lim, Kyu; Kim, Dong Woon; Choi, Dae Eun; Kim, Jwa-Jin

    2017-09-30

    Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI). Fat-1 transgenic mice produce ω3-Polyunsaturated fatty acids (ω3-PUFAs) from ω6-Polyunsaturated fatty acids (ω6-PUFAs) without a dietary ω3-PUFAs supplement, leading to a high accumulation of omega-3 in various tissues. ω3-PUFAs show protective effects against various renal injuries and it has recently been reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI) and evaluated its associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt) were divided into four groups: wt sham ( n = 10), fat-1 sham ( n = 10), wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15), and fat-1 IRI ( n = 15). Kidneys and blood were harvested 24 h after IRI and renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and Atg7; lower amounts of p62; and, higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more adenosine monophosphate-activated protein kinase (AMPK) activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR). Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.

  16. Nucleotide-oligomerizing domain-1 (NOD1) receptor activation induces pro-inflammatory responses and autophagy in human alveolar macrophages.

    Science.gov (United States)

    Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; Loyola, Elva; Escobedo, Dante; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Torres, Martha; Sada, Eduardo

    2014-09-25

    Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan fragments that localize to the cytosol. NOD1 activation triggers inflammation, antimicrobial mechanisms and autophagy in both epithelial cells and murine macrophages. NOD1 mediates intracellular pathogen clearance in the lungs of mice; however, little is known about NOD1's role in human alveolar macrophages (AMs) or its involvement in Mycobacterium tuberculosis (Mtb) infection. AMs, monocytes (MNs), and monocyte-derived macrophages (MDMs) from healthy subjects were assayed for NOD1 expression. Cells were stimulated with the NOD1 ligand Tri-DAP and cytokine production and autophagy were assessed. Cells were infected with Mtb and treated with Tri-DAP post-infection. CFUs counting determined growth control, and autophagy protein recruitment to pathogen localization sites was analyzed by immunoelectron microscopy. NOD1 was expressed in AMs, MDMs and to a lesser extent MNs. Tri-DAP stimulation induced NOD1 up-regulation and a significant production of IL1β, IL6, IL8, and TNFα in AMs and MDMs; however, the level of NOD1-dependent response in MNs was limited. Autophagy activity determined by expression of proteins Atg9, LC3, IRGM and p62 degradation was induced in a NOD1-dependent manner in AMs and MDMs but not in MNs. Infected AMs could be activated by stimulation with Tri-DAP to control the intracellular growth of Mtb. In addition, recruitment of NOD1 and the autophagy proteins IRGM and LC3 to the Mtb localization site was observed in infected AMs after treatment with Tri-DAP. NOD1 is involved in AM and MDM innate responses, which include proinflammatory cytokines and autophagy, with potential implications in the killing of Mtb in humans.

  17. Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

    International Nuclear Information System (INIS)

    Afeseh Ngwa, Hilary; Kanthasamy, Arthi; Gu, Yan; Fang, Ning; Anantharam, Vellareddy; Kanthasamy, Anumantha G.

    2011-01-01

    The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles (∼ 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25–400 μg/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase Cδ (PKCδ), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. -- Highlights: ► Mn nanoparticles activate mitochondrial cell death signaling

  18. Parkinson disease: a role for autophagy?

    Science.gov (United States)

    Yang, Qian; Mao, Zixu

    2010-08-01

    Autophagy is a term used to describe the process by which lysosomes degrade intracellular components. Known originally as an adaptive response to nutrient deprivation, autophagy has now been recognized to play important roles in several human disorders including neurodegenerative diseases. Experimental results from genetic, cellular, and toxicological studies indicate that many of the etiological factors associated with Parkinson disease (PD) can perturb the autophagic process in various model systems. Thus, the emerging data support the view that dysregulation of autophagy may play a critical role in the pathogenic process of PD.

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

    International Nuclear Information System (INIS)

    Malaviya, Rama; Laskin, Jeffrey D.; Laskin, Debra L.

    2014-01-01

    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

  20. 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.

  1. How Parkinsonian Toxins Dysregulate the Autophagy Machinery

    Directory of Open Access Journals (Sweden)

    Ruben K. Dagda

    2013-11-01

    Full Text Available Since their discovery, Parkinsonian toxins (6-hydroxydopamine, MPP+, paraquat, and rotenone have been widely employed as in vivo and in vitro chemical models of Parkinson’s disease (PD. Alterations in mitochondrial homeostasis, protein quality control pathways, and more recently, autophagy/mitophagy have been implicated in neurotoxin models of PD. Here, we highlight the molecular mechanisms by which different PD toxins dysregulate autophagy/mitophagy and how alterations of these pathways play beneficial or detrimental roles in dopamine neurons. The convergent and divergent effects of PD toxins on mitochondrial function and autophagy/mitophagy are also discussed in this review. Furthermore, we propose new diagnostic tools and discuss how pharmacological modulators of autophagy/mitophagy can be developed as disease-modifying treatments for PD. Finally, we discuss the critical need to identify endogenous and synthetic forms of PD toxins and develop efficient health preventive programs to mitigate the risk of developing PD.

  2. Intersection of autophagy with pathways of antigen presentation.

    Science.gov (United States)

    Patterson, Natalie L; Mintern, Justine D

    2012-12-01

    Traditionally, macroautophagy (autophagy) is viewed as a pathway of cell survival. Autophagy ensures the elimination of damaged or unwanted cytosolic components and provides a source of cellular nutrients during periods of stress. Interestingly, autophagy can also directly intersect with, and impact, other major pathways of cellular function. Here, we will review the contribution of autophagy to pathways of antigen presentation. The autophagy machinery acts to modulate both MHCI and MHCII antigen presentation. As such autophagy is an important participant in pathways that elicit host cell immunity and the elimination of infectious pathogens.

  3. SD118-Xanthocillin X (1, a Novel Marine Agent Extracted from Penicillium commune, Induces Autophagy through the Inhibition of the MEK/ERK Pathway

    Directory of Open Access Journals (Sweden)

    Caiguo Huang

    2012-06-01

    Full Text Available A compound named SD118-xanthocillin X (1 (C18H12N2O2, isolated from Penicillium commune in a deep-sea sediment sample, has been shown to inhibit the growth of several cancer cell lines in vitro. In the present study, we employed a growth inhibition assay and apoptotic analysis to identify the biological effect and detailed mechanism of SD118-xanthocillin X (1 in human hepatocellular carcinoma (HepG2 cells. SD118-xanthocillin X (1 demonstrated a concentration-dependent inhibitory effect on the growth of HepG2 cells and caused slight cellular apoptosis and significantly induced autophagy. Autophagy was detected as early as 12 h by the conversion of microtubule-associated protein 1 light chain 3 (LC3-I to LC3-II, following cleavage and lipid addition to LC3-I. The pharmacological autophagy inhibitor 3-methyladenine largely attenuates the growth inhibition and autophagic effect of SD118-xanthocillin X (1 in HepG2 cells. Our data also indicated that the autophagic effect of SD118-xanthocillin X (1 occurs via the down-regulation of the MEK/ERK signaling pathway and the up-regulated class III PI3K/Beclin 1 signaling pathway.

  4. Autophagy: More Than a Nonselective Pathway

    Directory of Open Access Journals (Sweden)

    Fulvio Reggiori

    2012-01-01

    Full Text Available Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs. For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy.

  5. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  6. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  7. The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

    Science.gov (United States)

    Kamitsuji, Y; Kuroda, J; Kimura, S; Toyokuni, S; Watanabe, K; Ashihara, E; Tanaka, H; Yui, Y; Watanabe, M; Matsubara, H; Mizushima, Y; Hiraumi, Y; Kawata, E; Yoshikawa, T; Maekawa, T; Nakahata, T; Adachi, S

    2008-11-01

    Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

  8. Cucurbitacin B inhibits proliferation, induces G2/M cycle arrest and autophagy without affecting apoptosis but enhances MTT reduction in PC12 cells

    Directory of Open Access Journals (Sweden)

    Chuanhong Wu

    2016-03-01

    Full Text Available In the present study, the effect of cucurbitacin B (a natural product with anti-cancer effect was studied on PC12 cells. It significantly reduced the cell number, changed cell morphology and inhibited colony formation while MTT results showed increased cell viability. Cucurbitacin B treatment increased activity of succinode hydrogenase. No alteration in the integrity of mem-brane, the release of lactic dehydrogenase, the mitochondrial membrane potential, and the expression of apoptotic proteins suggested that cucurbitacin B did not induce apoptosis. The cell cycle was remarkably arrested at G2/M phase. Furthermore, cucurbitacin B induced autophagy as evidence by accumulation of autophagic vacuoles and the increase of LC3II. In addition, cucurbitacin B up-regulated the expression of p-beclin-1, p-ULK1, p-Wee1, p21 and down-regulated p-mTOR, p-p70S6K, CDC25C, CDK1, Cyclin B1. In conclusion, cucurbitacin B inhibited PC12 proliferation but caused MTT pitfall. Cucurbitacin B induced G2/M cell cycle arrest, autophagy, but not the apoptosis in PC12 cells.

  9. Functional Analysis of the Beclin-1 Tumor Suppressor Interaction with hVps34 (Type-III P13'-kinase) in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Maltese, William A

    2008-01-01

    .... The lipid product of Vps34, PI(3)P, is required not only for autophagy, but also for assembly of proteins involved in endocytosis and trafficking of enzymes from the trans-Golgi network to the lysosomes...

  10. Agent-based modeling of autophagy reveals emergent regulatory behavior of spatio-temporal autophagy dynamics.

    Science.gov (United States)

    Börlin, Christoph S; Lang, Verena; Hamacher-Brady, Anne; Brady, Nathan R

    2014-09-10

    Autophagy is a vesicle-mediated pathway for lysosomal degradation, essential under basal and stressed conditions. Various cellular components, including specific proteins, protein aggregates, organelles and intracellular pathogens, are targets for autophagic degradation. Thereby, autophagy controls numerous vital physiological and pathophysiological functions, including cell signaling, differentiation, turnover of cellular components and pathogen defense. Moreover, autophagy enables the cell to recycle cellular components to metabolic substrates, thereby permitting prolonged survival under low nutrient conditions. Due to the multi-faceted roles for autophagy in maintaining cellular and organismal homeostasis and responding to diverse stresses, malfunction of autophagy contributes to both chronic and acute pathologies. We applied a systems biology approach to improve the understanding of this complex cellular process of autophagy. All autophagy pathway vesicle activities, i.e. creation, movement, fusion and degradation, are highly dynamic, temporally and spatially, and under various forms of regulation. We therefore developed an agent-based model (ABM) to represent individual components of the autophagy pathway, subcellular vesicle dynamics and metabolic feedback with the cellular environment, thereby providing a framework to investigate spatio-temporal aspects of autophagy regulation and dynamic behavior. The rules defining our ABM were derived from literature and from high-resolution images of autophagy markers under basal and activated conditions. Key model parameters were fit with an iterative method using a genetic algorithm and a predefined fitness function. From this approach, we found that accurate prediction of spatio-temporal behavior required increasing model complexity by implementing functional integration of autophagy with the cellular nutrient state. The resulting model is able to reproduce short-term autophagic flux measurements (up to 3

  11. ATP binding cassette G1-dependent cholesterol efflux during inflammation.

    Science.gov (United States)

    de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

    2011-02-01

    ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

  12. Regulation of Autophagy by Glucose in Mammalian Cells

    OpenAIRE

    Moruno, Félix; Pérez-Jiménez, Eva; Knecht, Erwin

    2012-01-01

    Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focu...

  13. Sorafenib-induced defective autophagy promotes cell death by necroptosis

    OpenAIRE

    Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Bj?rklund, Ann-Charlotte; Zhivotovsky, Boris; Grand?r, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

    2015-01-01

    Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencin...

  14. Functional Analysis of the Beclin-1 Tumor Suppressor Interaction with hVpa34 (Type III PI3’ -kinase) in Breast Cancer Cells

    Science.gov (United States)

    2005-06-01

    we subjected the cells to two established pro-autophagic stimuli; treatment with C2-ceramide ( Scarlatti et al., 2004) and nutrient deprivation...cell death ( Scarlatti et al., 2004;Yu et al., 2004;Shimizu et al., 2004). Nevertheless, without knowing if Beclin interaction with mVps34 is also...cathepsin D maturation. Biochem J. 353, 655-661. 29 Scarlatti ,F., Bauvy,C., Ventruti,A., Sala,G., Cluzeaud,F., Vandewalle,A., Ghidoni,R., and Codogno,P

  15. The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

    Science.gov (United States)

    Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

    2011-01-01

    The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their ‘fresh’ host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its ‘fresh’ host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. PMID:21729006

  16. Autophagy and Retromer Components in Plant Innate Immunity

    DEFF Research Database (Denmark)

    Munch, David

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

  17. Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages.

    Directory of Open Access Journals (Sweden)

    Toshihiko Suzuki

    2007-08-01

    Full Text Available Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD-like receptor (NLR family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC. We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

  18. Hyperosmotic stress stimulates autophagy via polycystin-2.

    Science.gov (United States)

    Peña-Oyarzun, Daniel; Troncoso, Rodrigo; Kretschmar, Catalina; Hernando, Cecilia; Budini, Mauricio; Morselli, Eugenia; Lavandero, Sergio; Criollo, Alfredo

    2017-08-22

    Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.

  19. A Molecular View of Autophagy in Lepidoptera

    Directory of Open Access Journals (Sweden)

    Davide Romanelli

    2014-01-01

    Full Text Available Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characterization of these pathways was undertaken only in recent years. In addition to developmentally programmed autophagy, there is growing interest in starvation-induced autophagy. Therefore we are now entering a new era of research on autophagy that foreshadows clarification of the role and regulatory mechanisms underlying this self-digesting process in Lepidoptera. Given that some of the most important lepidopteran species of high economic importance, such as the silkworm, Bombyx mori, belong to this insect order, we expect that this information on autophagy will be fully exploited not only in basic research but also for practical applications.

  20. Autophagy induction for the treatment of cancer.

    Science.gov (United States)

    Pietrocola, Federico; Pol, Jonathan; Vacchelli, Erika; Baracco, Elisa E; Levesque, Sarah; Castoldi, Francesca; Maiuri, Maria Chiara; Madeo, Frank; Kroemer, Guido

    2016-10-02

    Cancer can be viewed in 2 rather distinct ways, namely (i) as a cell-autonomous disease in which malignant cells have escaped control from cell-intrinsic barriers against proliferation and dissemination or (ii) as a systemic disease that involves failing immune control of aberrant cells. Since macroautophagy/autophagy generally increases the fitness of cells as well as their resistance against endogenous or iatrogenic (i.e., relating to illness due to medical intervention) stress, it has been widely proposed that inhibition of autophagy would constitute a valid strategy for sensitizing cancer cells to chemotherapy or radiotherapy. Colliding with this cell-autonomous vision, however, we found that immunosurveillance against transplantable, carcinogen-induced or genetically engineered cancers can be improved by pharmacologically inducing autophagy with caloric restriction mimetics. This positive effect depends on autophagy induction in cancer cells and is mediated by alterations in extracellular ATP metabolism, namely increased release of immunostimulatory ATP and reduced adenosine-dependent recruitment of immunosuppressive regulatory T cells into the tumor bed. The combination of autophagy inducers and chemotherapeutic agents is particularly efficient in reducing cancer growth through the stimulation of CD8 + T lymphocyte-dependent anticancer immune responses.

  1. 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.

  2. Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway.

    Science.gov (United States)

    Liu, Kuang-Kai; Qiu, Wei-Ru; Naveen Raj, Emmanuel; Liu, Huei-Fang; Huang, Hou-Syun; Lin, Yu-Wei; Chang, Chien-Jen; Chen, Ting-Hua; Chen, Chinpiao; Chang, Huan-Cheng; Hwang, Jenn-Kang; Chao, Jui-I

    2017-01-02

    Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

  3. Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

    International Nuclear Information System (INIS)

    Cerquetti, Lidia; Sampaoli, Camilla; Amendola, Donatella; Bucci, Barbara; Masuelli, Laura; Marchese, Rodolfo; Misiti, Silvia; De Venanzi, Agostino; Poggi, Maurizio; Toscano, Vincenzo; Stigliano, Antonio

    2011-01-01

    Thiazolidinediones, specific peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.

  4. Targeting the intracellular environment in cystic fibrosis: restoring autophagy as a novel strategy to circumvent the CFTR defect

    Directory of Open Access Journals (Sweden)

    Valeria Rachela Villella

    2013-01-01

    Full Text Available Cystic fibrosis (CF patients harboring the most common deletion mutation of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the plasma membrane even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1, a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the plasma membrane. We focus on the relationship between the improvement of peripheral proteostasis and CFTR plasma membrane stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent preclinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation.

  5. Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect

    Science.gov (United States)

    Villella, Valeria Rachela; Esposito, Speranza; Bruscia, Emanuela M.; Maiuri, Maria Chiara; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

    2013-01-01

    Cystic fibrosis (CF) patients harboring the most common deletion mutation of the CF transmembrane conductance regulator (CFTR), F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane (PM)-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the PM even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the PM. We focus on the relationship between the improvement of peripheral proteostasis and CFTR PM stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent pre-clinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation. PMID:23346057

  6. Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

    Science.gov (United States)

    Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

    2017-01-01

    Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

  7. Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cerquetti, Lidia; Sampaoli, Camilla [Endocrinology, Department of Clinical and Molecular Medicine, Sant' Andrea Hospital, Faculty of Medicine and Psychology ' Sapienza' University of Rome, Via di Grottarossa, 1035-00189 Rome (Italy); Research Center S. Pietro Hospital, Via Cassia, 600-00189 Rome (Italy); Amendola, Donatella; Bucci, Barbara [Research Center S. Pietro Hospital, Via Cassia, 600-00189 Rome (Italy); Masuelli, Laura [Department of Experimental Medicine, ' Sapienza' University of Rome, Rome (Italy); Marchese, Rodolfo [Research Center S. Pietro Hospital, Via Cassia, 600-00189 Rome (Italy); Misiti, Silvia [Endocrinology, Department of Clinical and Molecular Medicine, Sant' Andrea Hospital, Faculty of Medicine and Psychology ' Sapienza' University of Rome, Via di Grottarossa, 1035-00189 Rome (Italy); Research Center S. Pietro Hospital, Via Cassia, 600-00189 Rome (Italy); De Venanzi, Agostino; Poggi, Maurizio; Toscano, Vincenzo [Endocrinology, Department of Clinical and Molecular Medicine, Sant' Andrea Hospital, Faculty of Medicine and Psychology ' Sapienza' University of Rome, Via di Grottarossa, 1035-00189 Rome (Italy); Stigliano, Antonio, E-mail: antonio.stigliano@uniroma1.it [Endocrinology, Department of Clinical and Molecular Medicine, Sant' Andrea Hospital, Faculty of Medicine and Psychology ' Sapienza' University of Rome, Via di Grottarossa, 1035-00189 Rome (Italy); Research Center S. Pietro Hospital, Via Cassia, 600-00189 Rome (Italy)

    2011-06-10

    Thiazolidinediones, specific peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-{gamma}-inhibitor, showed that rosiglitazone acts through both PPAR-{gamma}-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-{gamma}. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPK{alpha} and beclin-1. The autophagy seems to be independent of PPAR-{gamma} activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.

  8. The Cytoskeleton-Autophagy Connection.

    Science.gov (United States)

    Kast, David J; Dominguez, Roberto

    2017-04-24

    Actin cytoskeleton dynamics play vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also have essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Branched actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Finally, mature autophagosomes detach from the ER membrane by an as yet unknown mechanism, undergo intracellular transport and then fuse with lysosomes, endosomes and multivesicular bodies through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins. In this review, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. Published by Elsevier Ltd.

  9. Autophagy in health and disease: focus on the cardiovascular system.

    Science.gov (United States)

    Mialet-Perez, Jeanne; Vindis, Cécile

    2017-12-12

    Autophagy is a highly conserved mechanism of lysosome-mediated protein and organelle degradation that plays a crucial role in maintaining cellular homeostasis. In the last few years, specific functions for autophagy have been identified in many tissues and organs. In the cardiovascular system, autophagy appears to be essential to heart and vessel homeostasis and function; however defective or excessive autophagy activity seems to contribute to major cardiovascular disorders including heart failure (HF) or atherosclerosis. Here, we review the current knowledge on the role of cardiovascular autophagy in physiological and pathophysiological conditions. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  10. Emerging role of autophagy in kidney function, diseases and aging

    Science.gov (United States)

    Huber, Tobias B.; Edelstein, Charles L.; Hartleben, Björn; Inoki, Ken; Jiang, Man; Koya, Daisuke; Kume, Shinji; Lieberthal, Wilfred; Pallet, Nicolas; Quiroga, Alejandro; Ravichandran, Kameswaran; Susztak, Katalin; Yoshida, Sei; Dong, Zheng

    2012-01-01

    Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy. PMID:22692002

  11. Autophagy in the light of sphingolipid metabolism

    DEFF Research Database (Denmark)

    Harvald, Eva Bang; Olsen, Anne Sofie Braun; Færgeman, Nils J.

    2015-01-01

    Maintenance of cellular homeostasis requires tight and coordinated control of numerous metabolic pathways, which are governed by interconnected networks of signaling pathways and energy-sensing regulators. Autophagy, a lysosomal degradation pathway by which the cell self-digests its own components......, has over the past decade been recognized as an essential part of metabolism. Autophagy not only rids the cell of excessive or damaged organelles, misfolded proteins, and invading microorganisms, it also provides nutrients to maintain crucial cellular functions. Besides serving as essential structural...... moieties of biomembranes, lipids including sphingolipids are increasingly being recognized as central regulators of a number of important cellular processes, including autophagy. In the present review we describe how sphingolipids, with special emphasis on ceramides and sphingosine-1-phosphate, can act...

  12. Induction of autophagy by spermidine promotes longevity.

    Science.gov (United States)

    Eisenberg, Tobias; Knauer, Heide; Schauer, Alexandra; Büttner, Sabrina; Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Ring, Julia; Schroeder, Sabrina; Magnes, Christoph; Antonacci, Lucia; Fussi, Heike; Deszcz, Luiza; Hartl, Regina; Schraml, Elisabeth; Criollo, Alfredo; Megalou, Evgenia; Weiskopf, Daniela; Laun, Peter; Heeren, Gino; Breitenbach, Michael; Grubeck-Loebenstein, Beatrix; Herker, Eva; Fahrenkrog, Birthe; Fröhlich, Kai-Uwe; Sinner, Frank; Tavernarakis, Nektarios; Minois, Nadege; Kroemer, Guido; Madeo, Frank

    2009-11-01

    Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

  13. IKK connects autophagy to major stress pathways.

    Science.gov (United States)

    Criollo, Alfredo; Senovilla, Laura; Authier, Hélène; Maiuri, Maria Chiara; Morselli, Eugenia; Vitale, Ilio; Kepp, Oliver; Tasdemir, Ezgi; Galluzzi, Lorenzo; Shen, Shensi; Tailler, Maximilien; Delahaye, Nicolas; Tesniere, Antoine; De Stefano, Daniela; Younes, Aména Ben; Harper, Francis; Pierron, Gérard; Lavandero, Sergio; Zitvogel, Laurence; Israel, Alain; Baud, Véronique; Kroemer, Guido

    2010-01-01

    Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

  14. Autophagy in breast cancer and its implications for therapy

    Science.gov (United States)

    Jain, Kirti; Paranandi, Krishna S; Sridharan, Savitha; Basu, Alakananda

    2013-01-01

    Autophagy is an evolutionarily conserved process of cellular self-digestion that serves as a mechanism to clear damaged organelles and recycle nutrients. Since autophagy can promote cell survival as well as cell death, it has been linked to different human pathologies, including cancer. Although mono-allelic deletion of autophagy-related gene BECN1 in breast tumors originally indicated a tumor suppressive role for autophagy in breast cancer, the intense research during the last decade suggests a role for autophagy in tumor progression. It is now recognized that tumor cells often utilize autophagy to survive various stresses, such as oncogene-induced transformation, hypoxia, endoplasmic reticulum (ER) stress and extracellular matrix detachment. Induction of autophagy by tumor cells may also contribute to tumor dormancy and resistance to anticancer therapies, thus making autophagy inhibitors promising drug candidates for breast cancer treatment. The scientific endeavors continue to define a precise role for autophagy in breast cancer. In this article, we review the current literature on the role of autophagy during the development and progression of breast cancer, and discuss the potential of autophagy modulators for breast cancer treatment. PMID:23841025

  15. Inhibition of autophagy initiation potentiates chemosensitivity in mesothelioma.

    Science.gov (United States)

    Follo, Carlo; Cheng, Yao; Richards, William G; Bueno, Raphael; Broaddus, Virginia Courtney

    2018-03-01

    The benefits of inhibiting autophagy in cancer are still controversial, with differences in outcome based on the type of tumor, the context and the particular stage of inhibition. Here, we investigated the impact of inhibiting autophagy at different stages on chemosensitivity using 3-dimensional (3D) models of mesothelioma, including ex vivo human tumor fragment spheroids. As shown by LC3B accumulation, we successfully inhibited autophagy using either an early stage ULK1/2 inhibitor (MRT 68921) or a late stage inhibitor (hydroxychloroquine). We found that inhibition of autophagy at the early stage, but not at late stage, potentiated chemosensitivity. This effect was seen only in those spheroids with high autophagy and active initiation at steady state. Inhibition of autophagy alone, at either early or late stage, did not cause cell death, showing that the inhibitors were non-toxic and that mesothelioma did not depend on autophagy at baseline, at least over 24 h. Using ATG13 puncta analysis, we found that autophagy initiation identified tumors that are more chemosensitive at baseline and after autophagy inhibition. Our results highlight a potential role of autophagy initiation in supporting mesothelioma cells during chemotherapy. Our work also highlights the importance of testing the inhibition of different stages in order to uncover the role of autophagy and the potential of its modulation in the treatment of cancer. © 2017 Wiley Periodicals, Inc.

  16. Polycystin-2-dependent control of cardiomyocyte autophagy.

    Science.gov (United States)

    Criollo, Alfredo; Altamirano, Francisco; Pedrozo, Zully; Schiattarella, Gabriele G; Li, Dan L; Rivera-Mejías, Pablo; Sotomayor-Flores, Cristian; Parra, Valentina; Villalobos, Elisa; Battiprolu, Pavan K; Jiang, Nan; May, Herman I; Morselli, Eugenia; Somlo, Stefan; de Smedt, Humbert; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2018-05-01

    Considerable evidence points to critical roles of intracellular Ca 2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca 2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca 2+ homeostasis and autophagy. Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2 F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca 2+ chelation using BAPTA-AM, whereas removal of extracellular Ca 2+ had no effect, pointing to a role of intracellular Ca 2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca 2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca 2+ -channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca 2+ . Furthermore, PC2 ablation was associated with impaired Ca 2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca 2+ stores. Finally, we provide evidence that Ca 2+ -mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. Together, this study unveils PC2 as a novel regulator of autophagy acting

  17. Autophagy: one more Nobel Prize for yeast

    Directory of Open Access Journals (Sweden)

    Andreas Zimmermann

    2016-12-01

    Full Text Available The recent announcement of the 2016 Nobel Prize in Physiology or Medicine, awarded to Yoshinori Ohsumi for the discoveries of mechanisms governing autophagy, underscores the importance of intracellular degradation and recycling. At the same time, it further cements yeast, in which this field decisively developed, as a prolific model organism. Here we provide a quick historical overview that mirrors both the importance of autophagy as a conserved and essential process for cellular life and death as well as the crucial role of yeast in its mechanistic characterization.

  18. 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......RNAs to regulation of the autophagy pathway. This regulation occurs both through specific core pathway components as well as through less well-defined mechanisms. Although this field is still in its infancy, we are beginning to understand the potential implications of these initial findings, both from a pathological...

  19. Molecular Interactions of Autophagy with the Immune System and Cancer

    Directory of Open Access Journals (Sweden)

    Yunho Jin

    2017-08-01

    Full Text Available Autophagy is a highly conserved catabolic mechanism that mediates the degradation of damaged cellular components by inducing their fusion with lysosomes. This process provides cells with an alternative source of energy for the synthesis of new proteins and the maintenance of metabolic homeostasis in stressful environments. Autophagy protects against cancer by mediating both innate and adaptive immune responses. Innate immune receptors and lymphocytes (T and B are modulated by autophagy, which represent innate and adaptive immune responses, respectively. Numerous studies have demonstrated beneficial roles for autophagy induction as well as its suppression of cancer cells. Autophagy may induce either survival or death depending on the cell/tissue type. Radiation therapy is commonly used to treat cancer by inducing autophagy in human cancer cell lines. Additionally, melatonin appears to affect cancer cell death by regulating programmed cell death. In this review, we summarize the current understanding of autophagy and its regulation in cancer.

  20. Role of autophagy in development and progression of acute pancreatitis

    Directory of Open Access Journals (Sweden)

    YANG Shuli

    2014-08-01

    Full Text Available Acute pancreatitis is considered an autodigestive disorder in which inappropriate activation of trypsinogen to trypsin within pancreatic acinar cells leads to the development of pancreatitis. Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, and it is one of the early pathological processes in acute pancreatitis. Autophagic flux is impaired in acute pancreatitis, which mediates the key pathologic responses of this disease. Impaired autophagy, dysfunction of lysosomes, and dysregulation of autophagy suggest a disorder of the endolysosomal pathway in acute pancreatitis. The role of autophagy in acute pancreatitis is discussed from the aspects of autophagic process, autophagy and activation of trypsinogen, impaired autophagy and acute pancreatitis, and defective autophagy promoting inflammation.

  1. Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats.

    Science.gov (United States)

    Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

    2015-01-01

    This study aimed to determine the effects of Bauhinia championii flavone (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore potential mechanisms. The MI/RI model in rats was established by ligating the left anterior descending coronary artery for 30 minutes, then reperfusing for 3 hours. BCF at 20 mg/kg was given 20 minutes prior to ischemia via sublingual intravenous injection, with 24 μg/kg phosphoinositide 3-kinase inhibitor (PI3K; wortmannin) as a control. The creatine kinase-MB and nitric oxide content were assessed by colorimetry. The levels of mitochondrial permeability transition pores and tumor necrosis factor alpha were determined by an enzyme-linked immunosorbent assay. Cardiomyocyte apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the expression of PI3K, endothelial nitric oxide synthase, caspase-3, and Beclin1 was analyzed by fluorescence quantitative polymerase chain reaction and Western blotting, respectively. Akt and microtubule-associated protein 1 light chain 3-II protein levels were also evaluated. Pretreatment with BCF significantly decreased the levels of creatine kinase-MB, tumor necrosis factor alpha, and mitochondrial permeability transition pores, but increased the nitric oxide content. Furthermore, BCF inhibited apoptosis, downregulated caspase-3, Beclin1, and microtubule-associated protein 1 light chain 3-II, upregulated PI3K, and increased the protein levels of phosphorylated Akt and endothelial nitric oxide synthase. However, all of the previously mentioned effects of BCF were blocked when BCF was coadministered with wortmannin. In conclusion, these observations indicated that BCF has cardioprotective effects against MI/RI by reducing cell apoptosis and excessive autophagy, which might be related to the activation of the PI3K/Akt signaling pathway.

  2. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    International Nuclear Information System (INIS)

    Maruyama, Yoko; Sou, Yu-Shin; Kageyama, Shun; Takahashi, Takao; Ueno, Takashi; Tanaka, Keiji; Komatsu, Masaaki; Ichimura, Yoshinobu

    2014-01-01

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs

  3. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yoko [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Sou, Yu-Shin; Kageyama, Shun [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Takahashi, Takao [Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Ueno, Takashi [Division of Proteomics and Biomolecular Science, Center for Biomedical Research Resources, Juntendo University Graduate School of Medicine, Tokyo 113-8421 (Japan); Tanaka, Keiji [Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Komatsu, Masaaki, E-mail: komatsu-ms@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Biochemistry, School of Medicine, Niigata University, Niigata 951-8510 (Japan); Ichimura, Yoshinobu, E-mail: ichimura-ys@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan)

    2014-03-28

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.

  4. CD4 T cell autophagy is integral to memory maintenance.

    Science.gov (United States)

    Murera, Diane; Arbogast, Florent; Arnold, Johan; Bouis, Delphine; Muller, Sylviane; Gros, Frédéric

    2018-04-13

    Studies of mice deficient for autophagy in T cells since thymic development, concluded that autophagy is integral to mature T cell homeostasis. Basal survival and functional impairments in vivo, limited the use of these models to delineate the role of autophagy during the immune response. We generated Atg5 f/f distal Lck (dLck)-cre mice, with deletion of autophagy only at a mature stage. In this model, autophagy deficiency impacts CD8 + T cell survival but has no influence on CD4 + T cell number and short-term activation. Moreover, autophagy in T cells is dispensable during early humoral response but critical for long-term antibody production. Autophagy in CD4 + T cells is required to transfer humoral memory as shown by injection of antigen-experienced cells in naive mice. We also observed a selection of autophagy-competent cells in the CD4 + T cell memory compartment. We performed in vitro differentiation of memory CD4 + T cells, to better characterize autophagy-deficient memory cells. We identified mitochondrial and lipid load defects in differentiated memory CD4 + T cells, together with a compromised survival, without any collapse of energy production. We then propose that memory CD4 + T cells rely on autophagy for their survival to regulate toxic effects of mitochondrial activity and lipid overload.

  5. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  6. Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging.

    Science.gov (United States)

    Plaza-Zabala, Ainhoa; Sierra-Torre, Virginia; Sierra, Amanda

    2017-03-09

    Autophagy is emerging as a core regulator of Central Nervous System (CNS) aging and neurodegeneration. In the brain, it has mostly been studied in neurons, where the delivery of toxic molecules and organelles to the lysosome by autophagy is crucial for neuronal health and survival. However, we propose that the (dys)regulation of autophagy in microglia also affects innate immune functions such as phagocytosis and inflammation, which in turn contribute to the pathophysiology of aging and neurodegenerative diseases. Herein, we first describe the basic concepts of autophagy and its regulation, discuss key aspects for its accurate monitoring at the experimental level, and summarize the evidence linking autophagy impairment to CNS senescence and disease. We focus on acute, chronic, and autoimmunity-mediated neurodegeneration, including ischemia/stroke, Alzheimer's, Parkinson's, and Huntington's diseases, and multiple sclerosis. Next, we describe the actual and potential impact of autophagy on microglial phagocytic and inflammatory function. Thus, we provide evidence of how autophagy may affect microglial phagocytosis of apoptotic cells, amyloid-β, synaptic material, and myelin debris, and regulate the progression of age-associated neurodegenerative diseases. We also discuss data linking autophagy to the regulation of the microglial inflammatory phenotype, which is known to contribute to age-related brain dysfunction. Overall, we update the current knowledge of autophagy and microglia, and highlight as yet unexplored mechanisms whereby autophagy in microglia may contribute to CNS disease and senescence.

  7. Impairment of autophagy: From hereditary disorder to drug intoxication

    International Nuclear Information System (INIS)

    Aki, Toshihiko; Funakoshi, Takeshi; Unuma, Kana; Uemura, Koichi

    2013-01-01

    At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinson's, Alzheimer's, and Huntington's diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication

  8. Deletion of protein tyrosine phosphatase 1B rescues against myocardial anomalies in high fat diet-induced obesity: Role of AMPK-dependent autophagy.

    Science.gov (United States)

    Kandadi, Machender R; Panzhinskiy, Evgeniy; Roe, Nathan D; Nair, Sreejayan; Hu, Dahai; Sun, Aijun

    2015-02-01

    Obesity-induced cardiomyopathy may be mediated by alterations in multiple signaling cascades involved in glucose and lipid metabolism. Protein tyrosine phosphatase-1B (PTP1B) is an important negative regulator of insulin signaling. This study was designed to evaluate the role of PTP1B in high fat diet-induced cardiac contractile anomalies. Wild-type and PTP1B knockout mice were fed normal (10%) or high (45%) fat diet for 5months prior to evaluation of cardiac function. Myocardial function was assessed using echocardiography and an Ion-Optix MyoCam system. Western blot analysis was employed to evaluate levels of AMPK, mTOR, raptor, Beclin-1, p62 and LC3-II. RT-PCR technique was employed to assess genes involved in hypertrophy and lipid metabolism. Our data revealed increased LV thickness and LV chamber size as well as decreased fractional shortening following high fat diet intake, the effect was nullified by PTP1B knockout. High fat diet intake compromised cardiomyocyte contractile function as evidenced by decreased peak shortening, maximal velocity of shortening/relengthening, intracellular Ca²⁺ release as well as prolonged duration of relengthening and intracellular Ca²⁺ decay, the effects of which were alleviated by PTP1B knockout. High fat diet resulted in enlarged cardiomyocyte area and increased lipid accumulation, which were attenuated by PTP1B knockout. High fat diet intake dampened myocardial autophagy as evidenced by decreased LC3-II conversion and Beclin-1, increased p62 levels as well as decreased phosphorylation of AMPK and raptor, the effects of which were significantly alleviated by PTP1B knockout. Pharmacological inhibition of AMPK using compound C disengaged PTP1B knockout-conferred protection against fatty acid-induced cardiomyocyte contractile anomalies. Taken together, our results suggest that PTP1B knockout offers cardioprotection against high fat diet intake through activation of AMPK. This article is part of a Special Issue entitled

  9. Wip1-dependent modulation of macrophage migration and phagocytosis

    DEFF Research Database (Denmark)

    Tang, Yiting; Pan, Bing; Zhou, Xin

    2017-01-01

    Macrophage accumulation within the vascular wall is a hallmark of atherosclerosis. Controlling macrophage conversion into foam cells remains a major challenge for treatment of atherosclerotic diseases. Here, we show that Wip1, a member of the PP2C family of Ser/Thr protein phosphatases, modulates...... macrophage migration and phagocytosis associated with atherosclerotic plaque formation. Wip1 deficiency increases migratory and phagocytic activities of the macrophage under stress conditions. Enhanced migration of Wip1-/- macrophages is mediated by Rac1-GTPase and PI3K/AKT signalling pathways. Elevated...... phagocytic ability of Wip1-/- macrophages is linked to CD36 plasma membrane recruitment that is regulated by AMPK activity. Our study identifies Wip1 as an intrinsic negative regulator of macrophage chemotaxis. We propose that Wip1-dependent control of macrophage function may provide avenues for preventing...

  10. Methods for assessing autophagy and autophagic cell death.

    Science.gov (United States)

    Tasdemir, Ezgi; Galluzzi, Lorenzo; Maiuri, M Chiara; Criollo, Alfredo; Vitale, Ilio; Hangen, Emilie; Modjtahedi, Nazanine; Kroemer, Guido

    2008-01-01

    Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

  11. Autophagy as a potential target for sarcoma treatment.

    Science.gov (United States)

    Min, Li; Choy, Edwin; Pollock, Raphael E; Tu, Chongqi; Hornicek, Francis; Duan, Zhenfeng

    2017-08-01

    Autophagy is a constitutively active, evolutionary conserved, catabolic process for maintaining homeostasis in cellular stress responses and cell survival. Although its mechanism has not been fully illustrated, recent work on autophagy in various types of sarcomas has demonstrated that autophagy exerts an important role in sarcoma cell growth and proliferation, in pro-survival response to therapies and stresses, and in therapeutic resistance of sarcoma. Thus, the autophagic process is being seen as a possibly novel therapeutic target of sarcoma. Additionally, some co-regulators of autophagy have also been investigated as promising biomarkers for the diagnosis and prognosis of sarcoma. In this review, we summarize contemporary advances in the role of autophagy in sarcoma and discuss the potential of autophagy as a new target for sarcoma treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Autophagy Facilitates Salmonella Replication in HeLa Cells

    Science.gov (United States)

    Yu, Hong B.; Croxen, Matthew A.; Marchiando, Amanda M.; Ferreira, Rosana B. R.; Cadwell, Ken; Foster, Leonard J.; Finlay, B. Brett

    2014-01-01

    ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. PMID:24618251

  13. Forms, Crosstalks, and the Role of Phospholipid Biosynthesis in Autophagy

    Directory of Open Access Journals (Sweden)

    Leanne Pereira

    2012-01-01

    Full Text Available Autophagy is a highly conserved cellular process occurring during periods of stress to ensure a cell's survival by recycling cytosolic constituents and making products that can be used in energy generation and other essential processes. Three major forms of autophagy exist according to the specific mechanism through which cytoplasmic material is transported to a lysosome. Chaperone-mediated autophagy is a highly selective form of autophagy that delivers specific proteins for lysosomal degradation. Microautophagy is a less selective form of autophagy that occurs through lysosomal membrane invaginations, forming tubes and directly engulfing cytoplasm. Finally, macroautophagy involves formation of new membrane bilayers (autophagosomes that engulf cytosolic material and deliver it to lysosomes. This review provides new insights on the crosstalks between different forms of autophagy and the significance of bilayer-forming phospholipid synthesis in autophagosomal membrane formation.

  14. Induction of autophagy is essential for monocyte-macrophage differentiation

    OpenAIRE

    Zhang, Yan; Morgan, Michael J.; Chen, Kun; Choksi, Swati; Liu, Zheng-gang

    2012-01-01

    Monocytes are programmed to undergo apoptosis in the absence of stimulation. Stimuli that promote monocyte-macrophage differentiation not only cause cellular changes, but also prevent the default apoptosis of monocytes. In the present study, we demonstrate that autophagy is induced when monocytes are triggered to differentiate and that the induction of autophagy is pivotal for the survival and differentiation of monocytes. We also show that inhibition of autophagy results in apoptosis of cell...

  15. Targeting Pediatric Glioma with Apoptosis and Autophagy Manipulation

    Science.gov (United States)

    2014-10-01

    that chloroquine treatments give the most reliable inhibition of autophagy without being directly cytotoxic. Bafilomycin can continue to be used for...in pediatric glioma and its interaction with RTK inhibition and apoptotic pathway activation will enable us to develop efficacious clinical trials...of autophagy, Rab7 and Lamp 2. We are now introducing siRNA against Rab7 and Lamp2 to reiterate the effects of Chloroquine inhibition of autophagy

  16. The Mucosal Immune System and Its Regulation by Autophagy.

    Science.gov (United States)

    Kabat, Agnieszka M; Pott, Johanna; Maloy, Kevin J

    2016-01-01

    The gastrointestinal tract presents a unique challenge to the mucosal immune system, which has to constantly monitor the vast surface for the presence of pathogens, while at the same time maintaining tolerance to beneficial or innocuous antigens. In the intestinal mucosa, specialized innate and adaptive immune components participate in directing appropriate immune responses toward these diverse challenges. Recent studies provide compelling evidence that the process of autophagy influences several aspects of mucosal immune responses. Initially described as a "self-eating" survival pathway that enables nutrient recycling during starvation, autophagy has now been connected to multiple cellular responses, including several aspects of immunity. Initial links between autophagy and host immunity came from the observations that autophagy can target intracellular bacteria for degradation. However, subsequent studies indicated that autophagy plays a much broader role in immune responses, as it can impact antigen processing, thymic selection, lymphocyte homeostasis, and the regulation of immunoglobulin and cytokine secretion. In this review, we provide a comprehensive overview of mucosal immune cells and discuss how autophagy influences many aspects of their physiology and function. We focus on cell type-specific roles of autophagy in the gut, with a particular emphasis on the effects of autophagy on the intestinal T cell compartment. We also provide a perspective on how manipulation of autophagy may potentially be used to treat mucosal inflammatory disorders.

  17. Glucocorticoids induce autophagy in rat bone marrow mesenchymal stem cells

    DEFF Research Database (Denmark)

    Wang, L.; Fan, J.; Lin, Y. S.

    2015-01-01

    Glucocorticoidinduced osteoporosis (GIOP) is a widespread clinical complication following glucocorticoid therapy. This irreversible damage to boneforming and resorbing cells is essential in the pathogenesis of osteoporosis. Autophagy is a physiological process involved in the regulation of cells...... and their responses to diverse stimuli, however, the role of autophagy in glucocorticoidinduced damage to bone marrow mesenchymal stem cells (BMSCs) remains unclear. The current study confirmed that glucocorticoid administration impaired the proliferation of BMSCs. Transmission electron microscopy...... that in response to glucocorticoid administration, induced autophagy aids to maintain proliferation and prevent apoptosis of BMSCs. Thus, it is hypothesized that autophagy may be a novel target in the treatment or prevention of osteoporosis....

  18. Physiological roles of Regulated Ire1 Dependent Decay

    Directory of Open Access Journals (Sweden)

    Dina S. Coelho

    2014-04-01

    Full Text Available Ire1 is an important transducer of the Unfolded Protein Response (UPR that is activated by the accumulation of misfolded proteins in the Endoplamic Reticulum (ER stress. Activated Ire1 mediates the splicing of an intron from the mRNA of Xbp1, causing a frame-shift during translation and introducing a new carboxyl domain in the Xbp1 protein, which only then becomes a fully functional transcription factor. Studies using cell culture systems demonstrated that Ire1 also promotes the degradation of mRNAs encoding mostly ER-targeted proteins, to reduce the load of incoming ER client proteins during ER stress. This process was called RIDD (regulated Ire1-dependent decay, but its physiological significance remained poorly characterized beyond cell culture systems. Here we review several recent studies that have highlighted the physiological roles of RIDD in specific biological paradigms, such as photoreceptor differentiation in Drosophila or mammalian liver and endocrine pancreas function. These studies demonstrate the importance of RIDD in tissues undergoing intense secretory function and highlight the physiologic role of RIDD during UPR activation in cells and organisms.

  19. BACE1-Dependent Neuregulin-1 Signaling: An Implication for Schizophrenia

    Directory of Open Access Journals (Sweden)

    Zhengrong Zhang

    2017-09-01

    Full Text Available Schizophrenia is a chronic psychiatric disorder with a lifetime prevalence of about 1% in the general population. Recent studies have shown that Neuregulin-1 (Nrg1 is a candidate gene for schizophrenia. At least 15 alternative splicing of NRG1 isoforms all contain an extracellular epidermal growth factor (EGF-like domain, which is sufficient for Nrg1 biological activity including the formation of myelin sheaths and the regulation of synaptic plasticity. It is known that Nrg1 can be cleaved by β-secretase (BACE1 and the resulting N-terminal fragment (Nrg1-ntf binds to receptor tyrosine kinase ErbB4, which activates Nrg1/ErbB4 signaling. While changes in Nrg1 expression levels in schizophrenia still remain controversial, understanding the BACE1-cleaved Nrg1-ntf and Nrg1/ErbB4 signaling in schizophrenia neuropathogenesis is essential and important. In this review paper, we included three major parts: (1 Nrg1 structure and cleavage pattern by BACE1; (2 BACE1-dependent Nrg1 cleavage associated with schizophrenia in human studies; and (3 Animal studies of Nrg1 and BACE1 mutations with behavioral observations. Our review will provide a better understanding of Nrg1 in schizophrenia and a potential strategy for using BACE1 cleavage of Nrg1 as a unique biomarker for diagnosis, as well as a new therapeutic target, of schizophrenia.

  20. Signaling induced by hop/STI-1 depends on endocytosis

    International Nuclear Information System (INIS)

    Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael

    2007-01-01

    The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP C ), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP C and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP C by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases

  1. Dysregulation of Autophagy Contributes to Anal Carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Evie H Carchman

    Full Text Available Autophagy is an intracellular catabolic process that removes and recycles unnecessary/dysfunctional cellular components, contributing to cellular health and survival. Autophagy is a highly regulated cellular process that responds to several intracellular signals, many of which are deregulated by human papillomavirus (HPV infection through the expression of HPV-encoded oncoproteins. This adaptive inhibitory response helps prevent viral clearance. A strong correlation remains between HPV infection and the development of squamous cell carcinoma (SCC of the anus, particularly in HIV positive and other immunosuppressed patients. We hypothesize that autophagy is inhibited by HPV-encoded oncoproteins thereby promoting anal carcinogenesis (Fig 1.HPV16 transgenic mice (K14E6/E7 and non-transgenic mice (FVB/N, both of which do not spontaneously develop anal tumors, were treated topically with the chemical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA, to induce anal cancer. The anuses at different time points of treatment (5, 10, 15 and 20 weeks were analyzed using immunofluorescence (IF for two key autophagy marker proteins (LC3β and p62 in addition to histological grading. The anuses from the K14E6/E7 mice were also analyzed for visual evidence of autophagic activity by electron microscopy (EM. To see if there was a correlation to humans, archival anal specimens were assessed histologically for grade of dysplasia and then analyzed for LC3β and p62 protein content. To more directly examine the effect of autophagic inhibition on anal carcinogenesis, nontransgenic mice that do not develop anal cancer with DMBA treatment were treated with a known pharmacologic inhibitor of autophagy, chloroquine, and examined for tumor development and analyzed by IF for autophagic proteins.Histologically, we observed the progression of normal anoderm to invasive SCC with DMBA treatment in K14E6/E7 mice but not in nontransgenic, syngeneic FVB/N background control mice

  2. Autophagy and phagocytosis converge for better vision.

    Science.gov (United States)

    Ferguson, Thomas A; Green, Douglas R

    2014-01-01

    The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.

  3. Quiltophagy--autophagy as folk art.

    Science.gov (United States)

    Crumrine, Barbara M; Klionsky, Daniel J

    2015-01-01

    Over the years macroautophagy (hereafter autophagy) has been depicted artistically through painting, music, dance, videos, and poetry. A unifying idea behind these different aesthetic approaches is that people learn in different ways. Thus, some learners may be engaged by a detailed, but static, painting, whereas others may find insight through the dynamic visualization provided by a dance. While each of these formats has advantages, they also have a common weakness--whether delivered through watercolor on a canvas, words on a paper, or movement captured in a video, they are all 2-dimensional. Yet, some people are tactile learners. In this paper, a quilter describes a project she created with the goal of demonstrating autophagy using a 3-dimensional approach, in which different fiber textures could be used to elaborate certain parts of the process.

  4. A Molecular View of Autophagy in Lepidoptera

    OpenAIRE

    Romanelli, Davide; Casati, Barbara; Franzetti, Eleonora; Tettamanti, Gianluca

    2014-01-01

    Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characteri...

  5. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

    Science.gov (United States)

    Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R; Sheng, Zhi

    2018-02-12

    Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

  6. β-Cell Autophagy in Diabetes Pathogenesis.

    Science.gov (United States)

    Marasco, Michelle R; Linnemann, Amelia K

    2018-05-01

    Nearly 100 years have passed since Frederick Banting and Charles Best first discovered and purified insulin. Their discovery and subsequent improvements revolutionized the treatment of diabetes, and the field continues to move at an ever-faster pace with respect to unique treatments for both type 1 and type 2 diabetes. Despite these advances, we still do not fully understand how apoptosis of the insulin-producing β-cells is triggered, presenting a challenge in the development of preventative measures. In recent years, the process of autophagy has generated substantial interest in this realm due to discoveries highlighting its clear role in the maintenance of cellular homeostasis. As a result, the number of studies focused on islet and β-cell autophagy has increased substantially in recent years. In this review, we will discuss what is currently known regarding the role of β-cell autophagy in type 1 and type 2 diabetes pathogenesis, with an emphasis on new and exciting developments over the past 5 years. Further, we will discuss how these discoveries might be translated into unique treatments in the coming years.

  7. Autophagy: a new player in skeletal maintenance?

    Science.gov (United States)

    Hocking, Lynne J; Whitehouse, Caroline; Helfrich, Miep H

    2012-07-01

    Imbalances between bone resorption and formation lie at the root of disorders such as osteoporosis, Paget's disease of bone (PDB), and osteopetrosis. Recently, genetic and functional studies have implicated proteins involved in autophagic protein degradation as important mediators of bone cell function in normal physiology and in pathology. Autophagy is the conserved process whereby aggregated proteins, intracellular pathogens, and damaged organelles are degraded and recycled. This process is important both for normal cellular quality control and in response to environmental or internal stressors, particularly in terminally-differentiated cells. Autophagic structures can also act as hubs for the spatial organization of recycling and synthetic process in secretory cells. Alterations to autophagy (reduction, hyperactivation, or impairment) are associated with a number of disorders, including neurodegenerative diseases and cancers, and are now being implicated in maintenance of skeletal homoeostasis. Here, we introduce the topic of autophagy, describe the new findings that are starting to emerge from the bone field, and consider the therapeutic potential of modifying this pathway for the treatment of age-related bone disorders. Copyright © 2012 American Society for Bone and Mineral Research.

  8. Autophagy in the eye: implications for ocular cell health.

    Science.gov (United States)

    Frost, Laura S; Mitchell, Claire H; Boesze-Battaglia, Kathleen

    2014-07-01

    Autophagy, a catabolic process by which a cell "eats" itself, turning over its own cellular constituents, plays a key role in cellular homeostasis. In an effort to maintain normal cellular function, autophagy is often up-regulated in response to environmental stresses and excessive organelle damage to facilitate aggregated protein removal. In the eye, virtually all cell types from those comprising the cornea in the front of the eye to the retinal pigment epithelium (RPE) providing a protective barrier for the retina at the back of the eye, rely on one or more aspects of autophagy to maintain structure and/or normal physiological function. In the lens autophagy plays a critical role in lens fiber cell maturation and the formation of the organelle free zone. Numerous studies delineating the role of Atg5, Vsp34 as well as FYCO1 in maintenance of lens transparency are discussed. Corneal endothelial dystrophies are also characterized as having elevated levels of autophagic proteins. Therefore, novel modulators of autophagy such as lithium and melatonin are proposed as new therapeutic strategies for this group of dystrophies. In addition, we summarize how corneal Herpes Simplex Virus (HSV-1) infection subverts the cornea's response to infection by inhibiting the normal autophagic response. Using glaucoma models we analyze the relative contribution of autophagy to cell death and cell survival. The cytoprotective role of autophagy is further discussed in an analysis of photoreceptor cell heath and function. We focus our analysis on the current understanding of autophagy in photoreceptor and RPE health, specifically on the diverse role of autophagy in rods and cones as well as its protective role in light induced degeneration. Lastly, in the RPE we highlight hybrid phagocytosis-autophagy pathways. This comprehensive review allows us to speculate on how alterations in various stages of autophagy contribute to glaucoma and retinal degenerations. Copyright © 2014 Elsevier Ltd

  9. Traditional Chinese Medicine CFF-1 induced cell growth inhibition, autophagy, and apoptosis via inhibiting EGFR-related pathways in prostate cancer.

    Science.gov (United States)

    Wu, Zhaomeng; Zhu, Qingyi; Yin, Yingying; Kang, Dan; Cao, Runyi; Tian, Qian; Zhang, Yu; Lu, Shan; Liu, Ping

    2018-04-01

    Traditional Chinese medicine (TCM) has a combined therapeutic result in cancer treatment by integrating holistic and local therapeutical effects, by which TCM can enhance the curative effect and reduce the side effect. In this study, we analyzed the effect of CFF-1 (alcohol extract from an anticancer compound Chinese medicine) on prostate cancer (PCa) cell lines and studied in detail the mechanism of cell death induced by CFF-1 in vitro and in vivo. From our data, we found for the first time that CFF-1 obviously arrested cell cycle in G1 phase, decreased cell viability and then increased nuclear rupture in a dose-dependent manner and finally resulted in apoptosis in prostate cancer cells. In molecular level, our data showed that CFF-1 induced inhibition of EGFR auto-phosphorylation and inactivation of EGFR. Disruption of EGFR activity in turn suppressed downstream PI3K/AKT and Raf/Erk signal pathways, resulted in the decrease of p-FOXO1 (Ser256) and regulated the expression of apoptosis-related and cycle-related genes. Moreover, CFF-1 markedly induced cell autophagy through inhibiting PI3K/AKT/mTOR pathway and then up-regulating Beclin-1 and LC-3II and down-regulating phosphorylation of p70S6K. In vivo, CFF-1-treated group exhibited a significant decrease in tumor volume compared with the negative control group in subcutaneous xenograft tumor in nude mice via inhibiting EGFR-related signal pathways. Thus, bio-functions of Chinese medicine CFF-1 in inducing PCa cell growth inhibition, autophagy, and apoptosis suggested that CFF-1 had the clinical potential to treat patients with prostate cancer. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  10. Preservation of autophagy should not direct nutritional therapy

    NARCIS (Netherlands)

    McClave, S.A.; Weijs, P.J.M.

    2015-01-01

    PURPOSE OF REVIEW: Recent reports in the literature have proposed that forced mandatory feeding should be avoided in the first week of critical illness to preserve autophagy, in order to maximize responses to oxidative stress, preserve organ function, and improve outcomes. RECENT FINDINGS: Autophagy

  11. Regulation of Autophagy by Glucose in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Erwin Knecht

    2012-07-01

    Full Text Available Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.

  12. Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol.

    Science.gov (United States)

    Morselli, Eugenia; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Maiuri, Maria Chiara; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2009-12-23

    Although autophagy has widely been conceived as a self-destructive mechanism that causes cell death, accumulating evidence suggests that autophagy usually mediates cytoprotection, thereby avoiding the apoptotic or necrotic demise of stressed cells. Recent evidence produced by our groups demonstrates that autophagy is also involved in pharmacological manipulations that increase longevity. Exogenous supply of the polyamine spermidine can prolong the lifespan of (while inducing autophagy in) yeast, nematodes and flies. Similarly, resveratrol can trigger autophagy in cells from different organisms, extend lifespan in nematodes, and ameliorate the fitness of human cells undergoing metabolic stress. These beneficial effects are lost when essential autophagy modulators are genetically or pharmacologically inactivated, indicating that autophagy is required for the cytoprotective and/or anti-aging effects of spermidine and resveratrol. Genetic and functional studies indicate that spermidine inhibits histone acetylases, while resveratrol activates the histone deacetylase Sirtuin 1 to confer cytoprotection/longevity. Although it remains elusive whether the same histones (or perhaps other nuclear or cytoplasmic proteins) act as the downstream targets of spermidine and resveratrol, these results point to an essential role of protein hypoacetylation in autophagy control and in the regulation of longevity.

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

    Science.gov (United States)

    Chaachouay, Hassan; Ohneseit, Petra; Toulany, Mahmoud; Kehlbach, Rainer; Multhoff, Gabriele; Rodemann, H Peter

    2011-06-01

    Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. VALSARTAN REGULATES MYOCARDIAL AUTOPHAGY AND MITOCHONDRIAL TURNOVER IN EXPERIMENTAL HYPERTENSION

    Science.gov (United States)

    Zhang, Xin; Li, Zi-Lun; Crane, John A.; Jordan, Kyra L.; Pawar, Aditya S.; Textor, Stephen C.; Lerman, Amir; Lerman, Lilach O.

    2014-01-01

    Renovascular hypertension alters cardiac structure and function. Autophagy is activated during left ventricular hypertrophy and linked to adverse cardiac function. The Angiotensin II receptor blocker Valsartan lowers blood pressure and is cardioprotective, but whether it modulates autophagy in the myocardium is unclear. We hypothesized that Valsartan would alleviate autophagy and improve left ventricular myocardial mitochondrial turnover in swine renovascular hypertension. Domestic pigs were randomized to control, unilateral renovascular hypertension, and renovascular hypertension treated with Valsartan (320 mg/day) or conventional triple therapy (Reserpine+hydralazine+hydrochlorothiazide) for 4 weeks post 6-weeks of renovascular hypertension (n=7 each group). Left ventricular remodeling, function and myocardial oxygenation and microcirculation were assessed by multi-detector computer tomography, blood-oxygen-level-dependent magnetic resonance imaging and microcomputer tomography. Myocardial autophagy, markers for mitochondrial degradation and biogenesis, and mitochondrial respiratory-chain proteins were examined ex vivo. Renovascular hypertension induced left ventricular hypertrophy and myocardial hypoxia, enhanced cellular autophagy and mitochondrial degradation, and suppressed mitochondrial biogenesis. Valsartan and triple therapy similarly decreased blood pressure, but Valsartan solely alleviated left ventricular hypertrophy, ameliorated myocardial autophagy and mitophagy, and increased mitochondrial biogenesis. In contrast, triple therapy only slightly attenuated autophagy and preserved mitochondrial proteins, but elicited no improvement in mitophagy. These data suggest a novel potential role of Valsartan in modulating myocardial autophagy and mitochondrial turnover in renovascular hypertension-induced hypertensive heart disease, which may possibly bolster cardiac repair via a blood pressure-independent manner. PMID:24752430

  15. Autophagy in Negative-Strand RNA Virus Infection

    Directory of Open Access Journals (Sweden)

    Yupeng Wang

    2018-02-01

    Full Text Available Autophagy is a homoeostatic process by which cytoplasmic material is targeted for degradation by the cell. Viruses have learned to manipulate the autophagic pathway to ensure their own replication and survival. Although much progress has been achieved in dissecting the interplay between viruses and cellular autophagic machinery, it is not well understood how the cellular autophagic pathway is utilized by viruses and manipulated to their own advantage. In this review, we briefly introduce autophagy, viral xenophagy and the interaction among autophagy, virus and immune response, then focus on the interplay between NS-RNA viruses and autophagy during virus infection. We have selected some exemplary NS-RNA viruses and will describe how these NS-RNA viruses regulate autophagy and the role of autophagy in NS-RNA viral replication and in immune responses to virus infection. We also review recent advances in understanding how NS-RNA viral proteins perturb autophagy and how autophagy-related proteins contribute to NS-RNA virus replication, pathogenesis and antiviral immunity.

  16. Autophagy-dependent secretion: contribution to tumor progression

    Directory of Open Access Journals (Sweden)

    Tom Keulers

    2016-11-01

    Full Text Available Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e. the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumour microenvironment and tumour progression. The autophagy mediated secreted factors may stimulate cellular proliferation via auto- and paracrine signaling. The autophagy mediated release of immune modulating proteins change the immunosuppresive tumor microenvironment and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking or alterations in homeostasis and/or autonomous cell signaling.

  17. Role of Autophagy in the Control of Body Metabolism

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    Wenying Quan

    2013-03-01

    Full Text Available Autophagy plays a crucial role in the maintenance of cellular nutrient balance and the function of organelles such as mitochondria or the endoplasmic reticulum, which are important in intracellular metabolism, insulin release, and insulin sensitivity. In the insulin-producing pancreatic β-cells, autophagy is important in the maintenance of β-cell mass, structure, and function. Mice with deficiencies in β-cell-specific autophagy show reduced β-cell mass and defects in insulin secretion that lead to hypoinsulinemia and hyperglycemia but not diabetes. However, these mice developed diabetes when bred with ob/ob mice, suggesting that autophagy-deficient β-cells have defects in dealing with the increased metabolic stress imposed by obesity. These results also imply that autophagy deficiency in β-cells could be a factor in the progression from obesity to diabetes. Another important function of autophagy is in hypothalamic neurons for the central control of energy expenditure, appetite, and body weight. In addition, mice with autophagy deficiencies in the target tissues of insulin have yielded diverse phenotypes. Taken together, these results suggest that autophagy is important in the control of whole body energy and nutrient homeostasis, and its dysregulation could play a role in the development of metabolic disorders and diabetes.

  18. The Nobel Prize for understanding autophagy, a cellular mechanism ...

    Indian Academy of Sciences (India)

    The lysosome was identified by Christian de Duve in the 1950s as a membrane bound organelle in thecell that contains degradative enzymes such as proteases, lipases, acid phosphatases, etc. (de Duve, 2005).The term autophagy was coined by Christian de Duve in 1963. Autophagy generally occurs at low level, butit ...

  19. Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

    Science.gov (United States)

    Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

    2017-09-01

    Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

  20. Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

    NARCIS (Netherlands)

    Folkerts, Hendrik; Hilgendorf, Susan; Wierenga, Albertus T J; Jaques, Jennifer; Mulder, André B; Coffer, Paul J; Schuringa, Jan Jacob; Vellenga, Edo

    2017-01-01

    Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34(+) AML cells with a large variability in basal autophagy between AMLs observed. The autophagy

  1. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    Science.gov (United States)

    Mulakkal, Nitha C.; Nagy, Peter; Takats, Szabolcs; Tusco, Radu; Juhász, Gábor; Nezis, Ioannis P.

    2014-01-01

    The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy. PMID:24949430

  2. Does autophagy work in synaptic plasticity and memory?

    Science.gov (United States)

    Shehata, Mohammad; Inokuchi, Kaoru

    2014-01-01

    Many studies have reported the roles played by regulated proteolysis in neural plasticity and memory. Within this context, most of the research focused on the ubiquitin-proteasome system and the endosome-lysosome system while giving lesser consideration to another major protein degradation system, namely, autophagy. Although autophagy intersects with many of the pathways known to underlie synaptic plasticity and memory, only few reports related autophagy to synaptic remodeling. These pathways include PI3K-mTOR pathway and endosome-dependent proteolysis. In this review, we will discuss several lines of evidence supporting a physiological role of autophagy in memory processes, and the possible mechanistic scenarios for how autophagy could fulfill this function.

  3. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    Directory of Open Access Journals (Sweden)

    Nitha C. Mulakkal

    2014-01-01

    Full Text Available The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy.

  4. Modulation of Autophagy-Like Processes by Tumor Viruses

    Directory of Open Access Journals (Sweden)

    Karl Munger

    2012-06-01

    Full Text Available Autophagy is an intracellular degradation pathway for long-lived proteins and organelles. This process is activated above basal levels upon cell intrinsic or environmental stress and dysregulation of autophagy has been linked to various human diseases, including those caused by viral infection. Many viruses have evolved strategies to directly interfere with autophagy, presumably to facilitate their replication or to escape immune detection. However, in some cases, modulation of autophagy appears to be a consequence of the virus disturbing the cell’s metabolic signaling networks. Here, we summarize recent advances in research at the interface of autophagy and viral infection, paying special attention to strategies that human tumor viruses have evolved.

  5. BECN2 interacts with ATG14 through a metastable coiled-coil to mediate autophagy: BECN2 CCD Structure and Interaction with ATG14

    Energy Technology Data Exchange (ETDEWEB)

    Su, Minfei; Li, Yue; Wyborny, Shane; Neau, David; Chakravarthy, Srinivas; Levine, Beth; Colbert, Christopher L.; Sinha, Sangita C. (NDSU); (IIT); (Cornell); (UTSMC)

    2017-03-12

    ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the α-helical, coiled-coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self-association and thermal stability. Unlike BECN1, all BECN2 CCD mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure-based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.

  6. Autophagy-related genes in Helicobacter pylori infection.

    Science.gov (United States)

    Tanaka, Shingo; Nagashima, Hiroyuki; Uotani, Takahiro; Graham, David Y; Yamaoka, Yoshio

    2017-06-01

    In vitro studies have shown that Helicobacter pylori (H. pylori) infection induces autophagy in gastric epithelial cells. However, prolonged exposure to H. pylori reduces autophagy by preventing maturation of the autolysosome. The alterations of the autophagy-related genes in H. pylori infection are not yet fully understood. We analyzed autophagy-related gene expression in H. pylori-infected gastric mucosa compared with uninfected gastric mucosa obtained from 136 Bhutanese volunteers with mild dyspeptic symptoms. We also studied single nucleotide polymorphisms (SNPs) of autophagy-related gene in 283 Bhutanese participants to identify the influence on susceptibility to H. pylori infection. Microarray analysis of 226 autophagy-related genes showed that 16 genes were upregulated (7%) and nine were downregulated (4%). We used quantitative reverse transcriptase polymerase chain reaction to measure mRNA levels of the downregulated genes (ATG16L1, ATG5, ATG4D, and ATG9A) that were core molecules of autophagy. ATG16L1 and ATG5 mRNA levels in H. pylori-positive specimens (n=86) were significantly less than those in H. pylori-negative specimens (n=50). ATG16L1 mRNA levels were inversely related to H. pylori density. We also compared SNPs of ATG16L1 (rs2241880) among 206 H. pylori-positive and 77 H. pylori-negative subjects. The odds ratio for the presence of H. pylori in the GG genotype was 0.40 (95% CI: 0.18-0.91) relative to the AA/AG genotypes. Autophagy-related gene expression profiling using high-throughput microarray analysis indicated that downregulation of core autophagy machinery genes may depress autophagy functions and possibly provide a better intracellular habit for H. pylori in gastric epithelial cells. © 2017 John Wiley & Sons Ltd.

  7. Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

    Directory of Open Access Journals (Sweden)

    Chih-Peng Chang

    Full Text Available Interferon-gamma (IFN-γ, a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1 translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/- mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

  8. The Scavenger Protein Apoptosis Inhibitor of Macrophages (AIM) Potentiates the Antimicrobial Response against Mycobacterium tuberculosis by Enhancing Autophagy

    Science.gov (United States)

    Sanjurjo, Lucía; Amézaga, Núria; Vilaplana, Cristina; Cáceres, Neus; Marzo, Elena; Valeri, Marta; Cardona, Pere-Joan; Sarrias, Maria-Rosa

    2013-01-01

    Apoptosis inhibitor of macrophages (AIM), a scavenger protein secreted by tissue macrophages, is transcriptionally regulated by the nuclear receptor Liver X Receptor (LXR) and Retinoid X Receptor (RXR) heterodimer. Given that LXR exerts a protective immune response against M. tuberculosis, here we analyzed whether AIM is involved in this response. In an experimental murine model of tuberculosis, AIM serum levels peaked dramatically early after infection with M. tuberculosis, providing an in vivo biological link to the disease. We therefore studied the participation of AIM in macrophage response to M. tuberculosis in vitro. For this purpose, we used the H37Rv strain to infect THP-1 macrophages transfected to stably express AIM, thereby increasing infected macrophage survival. Furthermore, the expression of this protein enlarged foam cell formation by enhancing intracellular lipid content. Phagocytosis assays with FITC-labeled M. tuberculosis bacilli indicated that this protein was not involved in bacterial uptake; however, AIM expression decreased the number of intracellular cfus by up to 70% in bacterial killing assays, suggesting that AIM enhances macrophage mycobactericidal activity. Accordingly, M. tuberculosis-infected AIM-expressing cells upregulated the production of reactive oxygen species. Moreover, real-time PCR analysis showed increased mRNA levels of the antimicrobial peptides cathelicidin and defensin 4B. These increases were concomitant with greater cellular concentrations of the autophagy-related molecules Beclin 1 and LC3II, as well as enhanced acidification of mycobacterial phagosomes and LC3 co-localization. In summary, our data support the notion that AIM contributes to key macrophage responses to M. tuberculosis. PMID:24223991

  9. HSF1 stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis

    Science.gov (United States)

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Taguchi, Katsutoshi; Tanaka, Masaki

    2017-01-01

    ABSTRACT Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis. PMID:27846364

  10. HSF1 stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis.

    Science.gov (United States)

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Taguchi, Katsutoshi; Tanaka, Masaki

    2017-01-02

    Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis.

  11. Canonical autophagy does not contribute to cellular radioresistance

    International Nuclear Information System (INIS)

    Schaaf, Marco B.E.; Jutten, Barry; Keulers, Tom G.; Savelkouls, Kim G.M.; Peeters, Hanneke J.M.; Beucken, Twan van den; Schooten, Frederik-Jan van; Godschalk, Roger W.; Vooijs, Marc; Rouschop, Kasper M.A.

    2015-01-01

    Background: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. Materials and methods: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. Results: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. Conclusion: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation

  12. Distinct Contributions of Autophagy Receptors in Measles Virus Replication.

    Science.gov (United States)

    Petkova, Denitsa S; Verlhac, Pauline; Rozières, Aurore; Baguet, Joël; Claviere, Mathieu; Kretz-Remy, Carole; Mahieux, Renaud; Viret, Christophe; Faure, Mathias

    2017-05-22

    Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO₂ and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.

  13. The IKK complex contributes to the induction of autophagy.

    Science.gov (United States)

    Criollo, Alfredo; Senovilla, Laura; Authier, Hélène; Maiuri, Maria Chiara; Morselli, Eugenia; Vitale, Ilio; Kepp, Oliver; Tasdemir, Ezgi; Galluzzi, Lorenzo; Shen, Shensi; Tailler, Maximilien; Delahaye, Nicolas; Tesniere, Antoine; De Stefano, Daniela; Younes, Aména Ben; Harper, Francis; Pierron, Gérard; Lavandero, Sergio; Zitvogel, Laurence; Israel, Alain; Baud, Véronique; Kroemer, Guido

    2010-02-03

    In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IkappaB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-kappaB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-kappaB or ablation of the p65/RelA NF-kappaB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-kappaB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-beta suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

  14. Laser stimulation can activate autophagy in HeLa cells

    International Nuclear Information System (INIS)

    Wang, Yisen; Hu, Minglie; Wang, Chingyue; Lan, Bei; Cao, Youjia; He, Hao

    2014-01-01

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca 2+ dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  15. Nanomaterials and Autophagy: New Insights in Cancer Treatment

    International Nuclear Information System (INIS)

    Panzarini, Elisa; Inguscio, Valentina; Tenuzzo, Bernardetta Anna; Carata, Elisabetta; Dini, Luciana

    2013-01-01

    Autophagy represents a cell’s response to stress. It is an evolutionarily conserved process with diversified roles. Indeed, it controls intracellular homeostasis by degradation and/or recycling intracellular metabolic material, supplies energy, provides nutrients, eliminates cytotoxic materials and damaged proteins and organelles. Moreover, autophagy is involved in several diseases. Recent evidences support a relationship between several classes of nanomaterials and autophagy perturbation, both induction and blockade, in many biological models. In fact, the autophagic mechanism represents a common cellular response to nanomaterials. On the other hand, the dynamic nature of autophagy in cancer biology is an intriguing approach for cancer therapeutics, since during tumour development and therapy, autophagy has been reported to trigger both an early cell survival and a late cell death. The use of nanomaterials in cancer treatment to deliver chemotherapeutic drugs and target tumours is well known. Recently, autophagy modulation mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anti-cancer agent. Herein, we summarize the effects of nanomaterials on autophagic processes in cancer, also considering the therapeutic outcome of synergism between nanomaterials and autophagy to improve existing cancer therapies

  16. Autophagy as an Emerging Common Pathomechanism in Inherited Peripheral Neuropathies

    Directory of Open Access Journals (Sweden)

    Mansour Haidar

    2017-05-01

    Full Text Available The inherited peripheral neuropathies (IPNs comprise a growing list of genetically heterogeneous diseases. With mutations in more than 80 genes being reported to cause IPNs, a wide spectrum of functional consequences is expected to follow this genotypic diversity. Hence, the search for a common pathomechanism among the different phenotypes has become the holy grail of functional research into IPNs. During the last decade, studies on several affected genes have shown a direct and/or indirect correlation with autophagy. Autophagy, a cellular homeostatic process, is required for the removal of cell aggregates, long-lived proteins and dead organelles from the cell in double-membraned vesicles destined for the lysosomes. As an evolutionarily highly conserved process, autophagy is essential for the survival and proper functioning of the cell. Recently, neuronal cells have been shown to be particularly vulnerable to disruption of the autophagic pathway. Furthermore, autophagy has been shown to be affected in various common neurodegenerative diseases of both the central and the peripheral nervous system including Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this review we provide an overview of the genes involved in hereditary neuropathies which are linked to autophagy and we propose the disruption of the autophagic flux as an emerging common pathomechanism. We also shed light on the different steps of the autophagy pathway linked to these genes. Finally, we review the concept of autophagy being a therapeutic target in IPNs, and the possibilities and challenges of this pathway-specific targeting.

  17. Autophagy in the immune response to tuberculosis: clinical perspectives.

    LENUS (Irish Health Repository)

    Ní Cheallaigh, C

    2011-06-01

    A growing body of evidence points to autophagy as an essential component in the immune response to tuberculosis. Autophagy is a direct mechanism of killing intracellular Mycobacterium tuberculosis and also acts as a modulator of proinflammatory cytokine secretion. In addition, autophagy plays a key role in antigen processing and presentation. Autophagy is modulated by cytokines; it is stimulated by T helper type 1 (Th1) cytokines such as tumour necrosis factor (TNF)-α and interferon (IFN)-γ, and is inhibited by the Th2 cytokines interleukin (IL)-4 and IL-13 and the anti-inflammatory cytokine IL-10. Vitamin D, via cathelicidin, can also induce autophagy, as can Toll-like receptor (TLR)-mediated signals. Autophagy-promoting agents, administered either locally to the lungs or systemically, could have a clinical application as adjunctive treatment of drug-resistant and drug-sensitive tuberculosis. Moreover, vaccines which effectively induce autophagy could be more successful in preventing acquisition or reactivation of latent tuberculosis.

  18. Laser stimulation can activate autophagy in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yisen; Hu, Minglie; Wang, Chingyue [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Lan, Bei; Cao, Youjia [Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin (China); He, Hao, E-mail: haohe@tju.edu.cn [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China)

    2014-10-27

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca{sup 2+} dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  19. The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

    Directory of Open Access Journals (Sweden)

    María Cristina Vanrell

    2017-11-01

    Full Text Available Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

  20. 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

  1. Oxidative Stress, Redox Signaling, and Autophagy: Cell Death Versus Survival

    Science.gov (United States)

    Navarro-Yepes, Juliana; Burns, Michaela; Anandhan, Annadurai; Khalimonchuk, Oleh; del Razo, Luz Maria; Quintanilla-Vega, Betzabet; Pappa, Aglaia; Panayiotidis, Mihalis I.

    2014-01-01

    Abstract Significance: The molecular machinery regulating autophagy has started becoming elucidated, and a number of studies have undertaken the task to determine the role of autophagy in cell fate determination within the context of human disease progression. Oxidative stress and redox signaling are also largely involved in the etiology of human diseases, where both survival and cell death signaling cascades have been reported to be modulated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent Advances: To date, there is a good understanding of the signaling events regulating autophagy, as well as the signaling processes by which alterations in redox homeostasis are transduced to the activation/regulation of signaling cascades. However, very little is known about the molecular events linking them to the regulation of autophagy. This lack of information has hampered the understanding of the role of oxidative stress and autophagy in human disease progression. Critical Issues: In this review, we will focus on (i) the molecular mechanism by which ROS/RNS generation, redox signaling, and/or oxidative stress/damage alter autophagic flux rates; (ii) the role of autophagy as a cell death process or survival mechanism in response to oxidative stress; and (iii) alternative mechanisms by which autophagy-related signaling regulate mitochondrial function and antioxidant response. Future Directions: Our research efforts should now focus on understanding the molecular basis of events by which autophagy is fine tuned by oxidation/reduction events. This knowledge will enable us to understand the mechanisms by which oxidative stress and autophagy regulate human diseases such as cancer and neurodegenerative disorders. Antioxid. Redox Signal. 21, 66–85. PMID:24483238

  2. 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.

  3. Huang-Lian-Jie-Du-Decotion induced protective autophagy against the injury of cerebral ischemia/reperfusion via MAPK-mTOR signaling pathway.

    Science.gov (United States)

    Wang, Peng-Ran; Wang, Jun-Song; Zhang, Chao; Song, Xing-Fang; Tian, Na; Kong, Ling-Yi

    2013-08-26

    Huang-Lian-Jie-Du-Decotion (HLJDD, Hwangryun-Hae-Dok-Decotion in Japan), an ancient antipyretic and detoxifying traditional Chinese medicine formula, was reported to have protective effect on ischemic stroke. To investigate the therapeutic effect of HLJDD on ischemic stroke and explore its mode of action. A model of ischemic stroke in the rat was established after transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Rats were assigned randomly to groups of control, sham, transient ischemia/reperfusion (I/R), and three treatment groups by HLJDD at 2.5, 5.0, 10.0mg/kg. The neurological deficit, the cerebral infarct size, morphology abnormality, biochemical parameters were examined, and the levels of relevant proteins were determined by immunoblotting analysis to evaluate the protective effects of HLJDD on ischemic stroke and explore the underlying mechanism. Compared with I/R group, HLJDD significantly ameliorated neurological deficit and histopathology changes, decreased infarct area, and restored the levels of biochemical indicators including nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD and glutathione peroxidase (GSH-PX). HLJDD also notably elevated the levels of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and other autophagy related genes (Atgs), promoted the activation of extracellular signal-regulated kinases (ERK), protein kinase B (Akt), 3-phosphoinositide-dependent kinase (PDK1), and inhibited the activation of mammalian target of rapamycin (mTOR), c-Jun N-terminal protein kinases (JNK), p38, phosphatase and tensin homolog (PTEN). HLJDD showed neuroprotective effects on ischemic stroke, at least in part to the induced protective autophagy via the regulation of mitogen-activated protein kinase (MAPK) signals. This Akt-independent protective autophagy is favorable in the treatment of stroke, avoiding unfavorable side

  4. Autophagy in HCV Infection: Keeping Fat and Inflammation at Bay

    Directory of Open Access Journals (Sweden)

    Tiziana Vescovo

    2014-01-01

    Full Text Available Hepatitis C virus (HCV infection is one of the main causes of chronic liver disease. Viral persistence and pathogenesis rely mainly on the ability of HCV to deregulate specific host processes, including lipid metabolism and innate immunity. Recently, autophagy has emerged as a cellular pathway, playing a role in several aspects of HCV infection. This review summarizes current knowledge on the molecular mechanisms that link the HCV life cycle with autophagy machinery. In particular, we discuss the role of HCV/autophagy interaction in dysregulating inflammation and lipid homeostasis and its potential for translational applications in the treatment of HCV-infected patients.

  5. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages

    DEFF Research Database (Denmark)

    Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man

    2018-01-01

    Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed...... with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes...... have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays...

  6. 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.

  7. Interplay between apoptosis and autophagy in colorectal cancer.

    Science.gov (United States)

    Qian, Hao-Ran; Shi, Zhao-Qi; Zhu, He-Pan; Gu, Li-Hu; Wang, Xian-Fa; Yang, Yi

    2017-09-22

    Autophagy and apoptosis are two pivotal mechanisms in mediating cell survival and death. Cross-talk of autophagy and apoptosis has been documented in the tumorigenesis and progression of cancer, while the interplay between the two pathways in colorectal cancer (CRC) has not yet been comprehensively summarized. In this study, we outlined the basis of apoptosis and autophagy machinery firstly, and then reviewed the recent evidence in cellular settings or animal studies regarding the interplay between them in CRC. In addition, several key factors that modulate the cross-talk between autophagy and apoptosis as well as its significance in clinical practice were discussed. Understanding of the interplay between the cell death mechanisms may benefit the translation of CRC treatment from basic research to clinical use.

  8. Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

    Directory of Open Access Journals (Sweden)

    Fouzi El Magraoui

    2015-10-01

    Full Text Available Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-receptors. Ubiquitination plays a central role in this process, because it regulates early signaling events during the induction of autophagy and is also used as a degradation-tag on the potential autophagic cargo protein. Here, we review how the ubiquitin-dependent steps of autophagy are balanced or counteracted by deubiquitination events. Moreover, we highlight the functional role of the corresponding deubiquitinating enzymes and discuss how they might be involved in the occurrence of cancer, neurodegenerative diseases or infection with pathogenic bacteria.

  9. Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities.

    Science.gov (United States)

    Menzies, Fiona M; Fleming, Angeleen; Caricasole, Andrea; Bento, Carla F; Andrews, Stephen P; Ashkenazi, Avraham; Füllgrabe, Jens; Jackson, Anne; Jimenez Sanchez, Maria; Karabiyik, Cansu; Licitra, Floriana; Lopez Ramirez, Ana; Pavel, Mariana; Puri, Claudia; Renna, Maurizio; Ricketts, Thomas; Schlotawa, Lars; Vicinanza, Mariella; Won, Hyeran; Zhu, Ye; Skidmore, John; Rubinsztein, David C

    2017-03-08

    Autophagy is a conserved pathway that delivers cytoplasmic contents to the lysosome for degradation. Here we consider its roles in neuronal health and disease. We review evidence from mouse knockout studies demonstrating the normal functions of autophagy as a protective factor against neurodegeneration associated with intracytoplasmic aggregate-prone protein accumulation as well as other roles, including in neuronal stem cell differentiation. We then describe how autophagy may be affected in a range of neurodegenerative diseases. Finally, we describe how autophagy upregulation may be a therapeutic strategy in a wide range of neurodegenerative conditions and consider possible pathways and druggable targets that may be suitable for this objective. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Autophagy as a Therapeutic Target in Cardiovascular Disease

    Science.gov (United States)

    Nemchenko, Andriy; Chiong, Mario; Turer, Aslan; Lavandero, Sergio; Hill, Joseph A.

    2011-01-01

    The epidemic of heart failure continues apace, and development of novel therapies with clinical efficacy has lagged. Now, important insights into the molecular circuitry of cardiovascular autophagy have raised the prospect that this cellular pathway of protein quality control may be a target of clinical relevance. Whereas basal levels of autophagy are required for cell survival, excessive levels – or perhaps distinct forms of autophagic flux – contribute to disease pathogenesis. Our challenge will be to distinguish mechanisms that drive adaptive versus maladaptive autophagy and to manipulate those pathways for therapeutic gain. Recent evidence suggests this may be possible. Here, we review the fundamental biology of autophagy and its role in a variety of forms of cardiovascular disease. We discuss ways in which this evolutionarily conserved catabolic mechanism can be manipulated, discuss studies presently underway in heart disease, and provide our perspective on where this exciting field may lead in the future. PMID:21723289

  11. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

    KAUST Repository

    Castagnetti, Francesco; Fiacco, Elisabetta; Imbriano, Carol; Latella, Lucia

    2017-01-01

    with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor

  12. Are mitochondrial reactive oxygen species required for autophagy?

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jianfei, E-mail: jjf73@pitt.edu [Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh (United States); Maeda, Akihiro; Ji, Jing [Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh (United States); Baty, Catherine J.; Watkins, Simon C. [Center for Biologic Imaging, Department of Cell Biology and Physiology, University of Pittsburgh (United States); Greenberger, Joel S. [Department of Radiation Oncology, University of Pittsburgh (United States); Kagan, Valerian E., E-mail: kagan@pitt.edu [Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh (United States)

    2011-08-19

    Highlights: {yields} Autophageal and apoptotic pathways were dissected in cytochrome c deficient cells. {yields} Staurosporine (STS)-induced autophagy was not accompanied by ROS generation. {yields} Autophagy was detectable in mitochondrial DNA deficient {rho}{sup 0} cells. {yields} Mitochondrial ROS are not required for the STS-induced autophagy in HeLa cells. -- Abstract: Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H{sub 2}O{sub 2} was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient {rho}{sup o} HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.

  13. Are mitochondrial reactive oxygen species required for autophagy?

    International Nuclear Information System (INIS)

    Jiang, Jianfei; Maeda, Akihiro; Ji, Jing; Baty, Catherine J.; Watkins, Simon C.; Greenberger, Joel S.; Kagan, Valerian E.

    2011-01-01

    Highlights: → Autophageal and apoptotic pathways were dissected in cytochrome c deficient cells. → Staurosporine (STS)-induced autophagy was not accompanied by ROS generation. → Autophagy was detectable in mitochondrial DNA deficient ρ 0 cells. → Mitochondrial ROS are not required for the STS-induced autophagy in HeLa cells. -- Abstract: Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H 2 O 2 was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ o HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.

  14. Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

    OpenAIRE

    El Magraoui, Fouzi; Reidick, Christina; Meyer, Hemut E.; Platta, Harald W.

    2015-01-01

    Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-recep...

  15. p53 and ARF: Unexpected players in autophagy

    OpenAIRE

    Balaburski, Gregor M.; Hontz, Robert D.; Murphy, Maureen E.

    2010-01-01

    p53 and ARF are well-established tumor suppressor proteins that function together in the negative regulation of cancer. Recently, both of these proteins were found to play surprising roles in autophagy. Autophagy (“self-eating”) is a critical response of eukaryotic cells to metabolic and other stress. During this process, portions of the cytosol are sequestered into characteristic double membrane vesicles that are delivered to the lysosome for degradation, leading to the release of free amino...

  16. p53-Mediated Molecular Control of Autophagy in Tumor Cells

    Directory of Open Access Journals (Sweden)

    Maria Mrakovcic

    2018-03-01

    Full Text Available Autophagy is an indispensable mechanism of the eukaryotic cell, facilitating the removal and renewal of cellular components and thereby balancing the cell’s energy consumption and homeostasis. Deregulation of autophagy is now regarded as one of the characteristic key features contributing to the development of tumors. In recent years, the suppression of autophagy in combination with chemotherapeutic treatment has been approached as a novel therapy in cancer treatment. However, depending on the type of cancer and context, interference with the autophagic machinery can either promote or disrupt tumorigenesis. Therefore, disclosure of the major signaling pathways that regulate autophagy and control tumorigenesis is crucial. To date, several tumor suppressor proteins and oncogenes have emerged as eminent regulators of autophagy whose depletion or mutation favor tumor formation. The mammalian cell “janitor” p53 belongs to one of these tumor suppressors that are most commonly mutated in human tumors. Experimental evidence over the last decade convincingly reports that p53 can act as either an activator or an inhibitor of autophagy depending on its subcellular localization and its mode of action. This finding gains particular significance as p53 deficiency or mutant variants of p53 that accumulate in the cytoplasm of tumor cells enable activation of autophagy. Accordingly, we recently identified p53 as a molecular hub that regulates autophagy and apoptosis in histone deacetylase inhibitor-treated uterine sarcoma cells. In light of this novel experimental evidence, in this review, we focus on p53 signaling as a mediator of the autophagic pathway in tumor cells.

  17. Blue-Print Autophagy: Potential for Cancer Treatment

    OpenAIRE

    Nadia Ruocco; Susan Costantini; Maria Costantini

    2016-01-01

    The marine environment represents a very rich source of biologically active compounds with pharmacological applications. This is due to its chemical richness, which is claiming considerable attention from the health science communities. In this review we give a general overview on the marine natural products involved in stimulation and inhibition of autophagy (a type of programmed cell death) linked to pharmacological and pathological conditions. Autophagy represents a complex multistep cellu...

  18. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-05-15

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

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

    International Nuclear Information System (INIS)

    Kawamata, Tomoko; Kamada, Yoshiaki; Suzuki, Kuninori; Kuboshima, Norihiro; Akimatsu, Hiroshi; Ota, Shinichi; Ohsumi, Mariko; Ohsumi, Yoshinori

    2005-01-01

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

  20. Overweight in elderly people induces impaired autophagy in skeletal muscle.

    Science.gov (United States)

    Potes, Yaiza; de Luxán-Delgado, Beatriz; Rodriguez-González, Susana; Guimarães, Marcela Rodrigues Moreira; Solano, Juan J; Fernández-Fernández, María; Bermúdez, Manuel; Boga, Jose A; Vega-Naredo, Ignacio; Coto-Montes, Ana

    2017-09-01

    Sarcopenia is the gradual loss of skeletal muscle mass, strength and quality associated with aging. Changes in body composition, especially in skeletal muscle and fat mass are crucial steps in the development of chronic diseases. We studied the effect of overweight on skeletal muscle tissue in elderly people without reaching obesity to prevent this extreme situation. Overweight induces a progressive protein breakdown reflected as a progressive withdrawal of anabolism against the promoted catabolic state leading to muscle wasting. Protein turnover is regulated by a network of signaling pathways. Muscle damage derived from overweight displayed by oxidative and endoplasmic reticulum (ER) stress induces inflammation and insulin resistance and forces the muscle to increase requirements from autophagy mechanisms. Our findings showed that failure of autophagy in the elderly deprives it to deal with the cell damage caused by overweight. This insufficiently efficient autophagy leads to an accumulation of p62 and NBR1, which are robust markers of protein aggregations. This impaired autophagy affects myogenesis activity. Depletion of myogenic regulatory factors (MRFs) without links to variations in myostatin levels in overweight patients suggest a possible reduction of satellite cells in muscle tissue, which contributes to declined muscle quality. This discovery has important implications that improve the understanding of aged-related atrophy caused by overweight and demonstrates how impaired autophagy is one of the main responsible mechanisms that aggravate muscle wasting. Therefore, autophagy could be an interesting target for therapeutic interventions in humans against muscle impairment diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

    Directory of Open Access Journals (Sweden)

    Stern Stephan T

    2012-06-01

    Full Text Available Abstract The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

  2. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    International Nuclear Information System (INIS)

    Orfali, Nina; McKenna, Sharon L.; Cahill, Mary R.; Gudas, Lorraine J.; Mongan, Nigel P.

    2014-01-01

    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

  3. Regulation of the autophagy protein LC3 by phosphorylation

    Science.gov (United States)

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  4. Autophagy as a Possible Underlying Mechanism of Nanomaterial Toxicity

    Directory of Open Access Journals (Sweden)

    Vanessa Cohignac

    2014-07-01

    Full Text Available The rapid development of nanotechnologies is raising safety concerns because of the potential effects of engineered nanomaterials on human health, particularly at the respiratory level. Since the last decades, many in vivo studies have been interested in the pulmonary effects of different classes of nanomaterials. It has been shown that some of them can induce toxic effects, essentially depending on their physico-chemical characteristics, but other studies did not identify such effects. Inflammation and oxidative stress are currently the two main mechanisms described to explain the observed toxicity. However, the exact underlying mechanism(s still remain(s unknown and autophagy could represent an interesting candidate. Autophagy is a physiological process in which cytoplasmic components are digested via a lysosomal pathway. It has been shown that autophagy is involved in the pathogenesis and the progression of human diseases, and is able to modulate the oxidative stress and pro-inflammatory responses. A growing amount of literature suggests that a link between nanomaterial toxicity and autophagy impairment could exist. In this review, we will first summarize what is known about the respiratory effects of nanomaterials and we will then discuss the possible involvement of autophagy in this toxicity. This review should help understand why autophagy impairment could be taken as a promising candidate to fully understand nanomaterials toxicity.

  5. The role of autophagy in microbial infection and immunity

    Directory of Open Access Journals (Sweden)

    Desai M

    2015-01-01

    Full Text Available Mayura Desai,1 Rong Fang,2 Jiaren Sun11Department of Microbiology and Immunology, 2Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX, USAAbstract: The autophagy pathway represents an evolutionarily conserved cell recycling process that is activated in response to nutrient deprivation and other stress signals. Over the years, it has been linked to an array of cellular functions. Equally, a wide range of cell-intrinsic, as well as extracellular, factors have been implicated in the induction of the autophagy pathway. Microbial infections represent one such factor that can not only activate autophagy through specific mechanisms but also manipulate the response to the invading microbe's advantage. Moreover, in many cases, particularly among viruses, the pathway has been shown to be intricately involved in the replication cycle of the pathogen. Conversely, autophagy also plays a role in combating the infection process, both through direct destruction of the pathogen and as one of the key mediating factors in the host defense mechanisms of innate and adaptive immunity. Further, the pathway also plays a role in controlling the pathogenesis of infectious diseases by regulating inflammation. In this review, we discuss various interactions between pathogens and the cellular autophagic response and summarize the immunological functions of the autophagy pathway.Keywords: autophagy, xenophagy, antiviral, antibacterial

  6. Comparative study of the effects of PM1-induced oxidative stress on autophagy and surfactant protein B and C expressions in lung alveolar type II epithelial MLE-12 cells.

    Science.gov (United States)

    Bai, Ru; Guan, Longfei; Zhang, Wei; Xu, Jinxia; Rui, Wei; Zhang, Fang; Ding, Wenjun

    2016-12-01

    There is a strong link between smaller air pollution particles and a range of serious health conditions. Thus, there is a need for understanding the impacts of airborne fine particulate matter (PM) with an aerodynamic diameter of PM1) on lung alveolar epithelial cells. In the present study, mouse lung epithelial type II cell MLE-12 cells were used to examine the intracellular oxidative responses and the surfactant protein expressions after exposure to various concentrations of PM1 collected from an urban site and a steel-factory site (referred as uPM1 and sPM1 hereafter, respectively). Physicochemical characterization of PM1 was performed by using scanning electron microscopy and transmission electron microscopy. Cytotoxicity and autophagy induced by PM1 were assessed by using comprehensive approaches after MLE-12 cells were exposed to different concentrations of PM1 for various times. Expression of surfactant proteins B and C in MLE-12 cells was determined by Western blotting. All of the tested PM1 induced cytotoxicity evidenced by significant decrease of cell viability and increase of lactate dehydrogenase (LDH) release in a time- and concentration-dependent manner in the exposed cells compared with the unexposed cells. A similar pattern of increase of intercellular reactive oxygen species (ROS) generation and decrease of superoxide dismutase (SOD) and catalase (CAT) activities was also observed. PM1-induced autophagy was evidenced by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and increased levels of beclin1. Data from Western blotting showed significant decrease of surfactant protein B and C expressions. Relatively high concentrations of transition metals, including Fe, Cu and Mn, may be responsible for the higher toxicity of sPM1 compared with uPM1. Moreover, pretreatment with N-acetylcysteine (NAC) or Chelex (a metal chelating agent, which removes a large suite of metals from PM1) prevented the increase of

  7. Role of autophagy in disease resistance and hypersensitive response-associated cell death

    DEFF Research Database (Denmark)

    Hofius, Daniel; Munch, David; Bressendorff, Simon

    2011-01-01

    Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum...

  8. Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells.

    Science.gov (United States)

    Ren, Lipeng; Yang, Hong; Cui, Yanhua; Xu, Shuanshuan; Sun, Fen; Tian, Na; Hua, Jinlian; Peng, Sha

    2017-07-01

    Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active β-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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.

  10. The Effects of Kaempferol-Inhibited Autophagy on Osteoclast Formation.

    Science.gov (United States)

    Kim, Chang-Ju; Shin, Sang-Hun; Kim, Bok-Joo; Kim, Chul-Hoon; Kim, Jung-Han; Kang, Hae-Mi; Park, Bong-Soo; Kim, In-Ryoung

    2018-01-02

    Kaempferol, a flavonoid compound, is derived from the rhizome of Kaempferia galanga L ., which is used in traditional medicine in Asia. Autophagy has pleiotropic functions that are involved in cell growth, survival, nutrient supply under starvation, defense against pathogens, and antigen presentation. There are many studies dealing with the inhibitory effects of natural flavonoids in bone resorption. However, no studies have explained the relationship between the autophagic and inhibitory processes of osteoclastogenesis by natural flavonoids. The present study was undertaken to investigate the inhibitory effects of osteoclastogenesis through the autophagy inhibition process stimulated by kaempferol in murin macrophage (RAW 264.7) cells. The cytotoxic effect of Kaempferol was investigated by MTT assay. The osteoclast differentiation and autophagic process were confirmed via tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, western blot, and real-time PCR. Kaempferol controlled the expression of autophagy-related factors and in particular, it strongly inhibited the expression of p62/SQSTM1. In the western blot and real time-PCR analysis, when autophagy was suppressed with the application of 3-Methyladenine (3-MA) only, osteoclast and apoptosis related factors were not significantly affected. However, we found that after cells were treated with kaempferol, these factors inhibited autophagy and activated apoptosis. Therefore, we presume that kaempferol-inhibited autophagy activated apoptosis by degradation of p62/SQSTM1. Further study of the p62/SQSTM1 gene as a target in the autophagy mechanism, may help to delineate the potential role of kaempferol in the treatment of bone metabolism disorders.

  11. 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.

  12. Distinct patterns of autophagy evoked by two benzoxazine derivatives in vascular endothelial cells.

    Science.gov (United States)

    Wang, Li; Dong, ZhiWu; Huang, Bin; Zhao, BaoXiang; Wang, Hua; Zhao, Jing; Kung, HsiangFu; Zhang, ShangLi; Miao, JunYing

    2010-11-01

    Macroautophagy (referred to as autophagy) is an evolutionarily conserved, bulk-destruction process in eukaryotes. During this process, the cytoplasm containing long-lived proteins and organelles is engulfed into double-membrane autophagosomes, and ultimately undergoes enzymatic degradation within lysosomes. Autophagy serves as a prosurvival machinery, or it may contribute to cell death. Accumulating evidence indicates that autophagy is involved in the pathogenesis and intervention of various human diseases. Pharmacological autophagy modulators are arousing interest from biologists and clinical physicians in light of their potential for disease therapy and increasing our understanding of the mechanism of autophagy. In this study, we identified two autophagy enhancers, 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) and 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO), in human umbilical vein endothelial cells (HUVEC s) by autophagy assays, and demonstrate that ABO and DBO could stimulate autophagy in an mtor-independent and mtor-dependent manner, respectively; ABO-stimulated autophagy was attributed to the elevation of the Ca2+ channel annexin A7 (ANXA7), whereas DBO's effect was due to the level of intracellular reactive oxygen species (ROS). Importantly, we found that ANXA7 was essential for autophagy induction via modulating the intracellular calcium concentration ([Ca2+]i) in HUVEC s. In summary, our work introduced two distinct autophagy enhancers and highlighted the critical role of ANXA7 in endothelial autophagy.

  13. Targeting autophagy in obesity: from pathophysiology to management.

    Science.gov (United States)

    Zhang, Yingmei; Sowers, James R; Ren, Jun

    2018-04-23

    Obesity poses a severe threat to human health, including the increased prevalence of hypertension, insulin resistance, diabetes mellitus, cancer, inflammation, sleep apnoea and other chronic diseases. Current therapies focus mainly on suppressing caloric intake, but the efficacy of this approach remains poor. A better understanding of the pathophysiology of obesity will be essential for the management of obesity and its complications. Knowledge gained over the past three decades regarding the aetiological mechanisms underpinning obesity has provided a framework that emphasizes energy imbalance and neurohormonal dysregulation, which are tightly regulated by autophagy. Accordingly, there is an emerging interest in the role of autophagy, a conserved homeostatic process for cellular quality control through the disposal and recycling of cellular components, in the maintenance of cellular homeostasis and organ function by selectively ridding cells of potentially toxic proteins, lipids and organelles. Indeed, defects in autophagy homeostasis are implicated in metabolic disorders, including obesity, insulin resistance, diabetes mellitus and atherosclerosis. In this Review, the alterations in autophagy that occur in response to nutrient stress, and how these changes alter the course of obesogenesis and obesity-related complications, are discussed. The potential of pharmacological modulation of autophagy for the management of obesity is also addressed.

  14. The Role of Autophagy in the Pathogenesis of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Kosuke Yamahara

    2013-01-01

    Full Text Available Diabetic nephropathy is a leading cause of end-stage renal disease worldwide. The multipronged drug approach targeting blood pressure and serum levels of glucose, insulin, and lipids fails to fully prevent the onset and progression of diabetic nephropathy. Therefore, a new therapeutic target to combat diabetic nephropathy is required. Autophagy is a catabolic process that degrades damaged proteins and organelles in mammalian cells and plays a critical role in maintaining cellular homeostasis. The accumulation of proteins and organelles damaged by hyperglycemia and other diabetes-related metabolic changes is highly associated with the development of diabetic nephropathy. Recent studies have suggested that autophagy activity is altered in both podocytes and proximal tubular cells under diabetic conditions. Autophagy activity is regulated by both nutrient state and intracellular stresses. Under diabetic conditions, an altered nutritional state due to nutrient excess may interfere with the autophagic response stimulated by intracellular stresses, leading to exacerbation of organelle dysfunction and diabetic nephropathy. In this review, we discuss new findings showing the relationships between autophagy and diabetic nephropathy and suggest the therapeutic potential of autophagy in diabetic nephropathy.

  15. Autophagy: not good OR bad, but good AND bad.

    Science.gov (United States)

    Altman, Brian J; Rathmell, Jeffrey C

    2009-05-01

    Autophagy is a well-established mechanism to degrade intracellular components and provide a nutrient source to promote survival of cells in metabolic distress. Such stress can be caused by a lack of available nutrients or by insufficient rates of nutrient uptake. Indeed, growth factor deprivation leads to internalization and degradation of nutrient transporters, leaving cells with limited means to access extracellular nutrients even when plentiful.This loss of growth factor signaling and extracellular nutrients ultimately leads to apoptosis, but also activates autophagy, which may degrade intracellular components and provide fuel for mitochondrial bioenergetics. The precise metabolic role of autophagy and how it intersects with the apoptotic pathways in growth factor withdrawal, however, has been uncertain. Our recent findings ingrowth factor-deprived hematopoietic cells show that autophagy can simultaneously contribute to cell metabolism and initiate a pathway to sensitize cells to apoptotic death. This pathway may promote tissue homeostasis by ensuring that only cells with high resistance to apoptosis may utilize autophagy as a survival mechanism when growth factors are limiting and nutrient uptake decreases.

  16. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

    KAUST Repository

    Castagnetti, Francesco

    2017-06-13

    Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

  17. Regorafenib delays the proliferation of hepatocellular carcinoma by inducing autophagy.

    Science.gov (United States)

    Han, Rui; Li, Shixin

    2018-04-02

    The aim of the present study was to investigate the effects of regorafenib on hepatocellular carcinoma autophagy, thereby supressing the malignancy of HCC. First, HepG2 and Hep3B cell autophagy was investigated using GFP-LC3 transfection after the treatment of regorafenib. Then, the activation of Akt/mTOR signaling was analyzed using western blot. Our data showed that liver cancer cell autophagy was significantly induced by 20 μM regorafenib using GFP-LC3 transfection. Meanwhile, regorafenib-induced cell death could largely be abolished by 3-MA or CQ treatment, suggesting that regorafenib-induced HepG2 cell death was partially dependent on autophagy. Moreover, the activation of Akt/mTOR signaling was inhibited by regorafenib pre-incubation. MTT assay showed the combination use of regorafenib and CDDP led to a stronger growth inhibitory effect on HepG2 and Hep3B cells. In summary, regorafenib may acts an adjunctive therapy for liver cancer patients via modulating autophagy-dependent cell death even when apoptosis resistance is induced in cancer cells.

  18. Staying young at heart: autophagy and adaptation to cardiac aging.

    Science.gov (United States)

    Leon, Leonardo J; Gustafsson, Åsa B

    2016-06-01

    Aging is a predominant risk factor for developing cardiovascular disease. Therefore, the cellular processes that contribute to aging are attractive targets for therapeutic interventions that can delay or prevent the development of age-related diseases. Our understanding of the underlying mechanisms that contribute to the decline in cell and tissue functions with age has greatly advanced over the past decade. Classical hallmarks of aging cells include increased levels of reactive oxygen species, DNA damage, accumulation of dysfunctional organelles, oxidized proteins and lipids. These all contribute to a progressive decline in the normal physiological function of the cell and to the onset of age-related conditions. A major cause of the aging process is progressive loss of cellular quality control. Autophagy is an important quality control pathway and is necessary to maintain cardiac homeostasis and to adapt to stress. A reduction in autophagy has been observed in a number of aging models and there is compelling evidence that enhanced autophagy delays aging and extends life span. Enhancing autophagy counteracts age-associated accumulation of protein aggregates and damaged organelles in cells. In this review, we discuss the functional role of autophagy in maintaining homeostasis in the heart, and how a decline is associated with accelerated cardiac aging. We also evaluate therapeutic approaches being researched in an effort to maintain a healthy young heart. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. 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

    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. PMID:27077655

  20. Methods to Monitor and Manipulate TFEB Activity During Autophagy.

    Science.gov (United States)

    Medina, D L; Settembre, C; Ballabio, A

    2017-01-01

    Macroautophagy is a catabolic process deputed to the turnover of intracellular components. Recent studies have revealed that transcriptional regulation is a major mechanism controlling autophagy. Currently, more than 20 transcription factors have been shown to modulate cellular autophagy levels. Among them, the transcription factor EB (TFEB) appears to have the broadest proautophagy role, given its capacity to control the biogenesis of lysosomes and autophagosomes, the two main organelles required for the autophagy pathway. TFEB has attracted major attention owing to its ability to enhance cellular clearance of pathogenic substrates in a variety of animal models of disease, such as lysosomal storage disorders, Parkinson's, Alzheimer's, α1-antitrypsin, obesity as well as others, suggesting that the TFEB pathway represents an extraordinary possibility for future development of innovative therapies. Importantly, the subcellular localization and activity of TFEB are regulated by its phosphorylation status, suggesting that TFEB activity can be pharmacologically targeted. Given the growing list of common and rare diseases in which manipulation of autophagy may be beneficial, in this chapter we describe a set of validated protocols developed to modulate and analyze TFEB-mediated enhancement of autophagy both in vitro and in vivo conditions. © 2017 Elsevier Inc. All rights reserved.

  1. Enhanced Autophagy in Polycystic Kidneys of AQP11 Null Mice

    Directory of Open Access Journals (Sweden)

    Yasuko Tanaka

    2016-11-01

    Full Text Available Aquaporin-11 (AQP11 is an intracellular water channel expressed at the endoplasmic reticulum (ER of the proximal tubule. Its gene disruption in mice leads to intracellular vacuole formation at one week and the subsequent development of polycystic kidneys by three weeks. As the damaged proximal tubular cells with intracellular vacuoles form cysts later, we postulated that autophagy may play a role in the cyst formation and examined autophagy activity before and after cyst development in AQP11(−/− kidneys. PCR analysis showed the increased expression of the transcript encoding LC3 (Map1lc3b as well as other autophagy-related genes in AQP11(−/− mice. Using green fluorescent protein (GFP-LC3 transgenic mice and AQP11(−/− mice, we found that the number of GFP-LC3–positive puncta was increased in the proximal tubule of AQP11(−/− mice before the cyst formation. Interestingly, they were also observed in the cyst-lining epithelial cell. Further PCR analyses revealed the enhanced expression of apoptosis-related and ER stress–related caspase genes before and after the cyst formation, which may cause the enhanced autophagy. These results suggest the involvement of autophagy in the development and maintenance of kidney cysts in AQP11(−/− mice.

  2. Pollination induces autophagy in petunia petals via ethylene.

    Science.gov (United States)

    Shibuya, Kenichi; Niki, Tomoko; Ichimura, Kazuo

    2013-02-01

    Autophagy is one of the main mechanisms of degradation and remobilization of macromolecules, and it appears to play an important role in petal senescence. However, little is known about the regulatory mechanisms of autophagy in petal senescence. Autophagic processes were observed by electron microscopy and monodansylcadaverine staining of senescing petals of petunia (Petunia hybrida); autophagy-related gene 8 (ATG8) homologues were isolated from petunia and the regulation of expression was analysed. Nutrient remobilization was also examined during pollination-induced petal senescence. Active autophagic processes were observed in the mesophyll cells of senescing petunia petals. Pollination induced the expression of PhATG8 homologues and was accompanied by an increase in ethylene production. Ethylene inhibitor treatment in pollinated flowers delayed the induction of PhATG8 homologues, and ethylene treatment rapidly upregulated PhATG8 homologues in petunia petals. Dry weight and nitrogen content were decreased in the petals and increased in the ovaries after pollination in detached flowers. These results indicated that pollination induces autophagy and that ethylene is a key regulator of autophagy in petal senescence of petunia. The data also demonstrated the translocation of nutrients from the petals to the ovaries during pollination-induced petal senescence.

  3. Blue-Print Autophagy: Potential for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Nadia Ruocco

    2016-07-01

    Full Text Available The marine environment represents a very rich source of biologically active compounds with pharmacological applications. This is due to its chemical richness, which is claiming considerable attention from the health science communities. In this review we give a general overview on the marine natural products involved in stimulation and inhibition of autophagy (a type of programmed cell death linked to pharmacological and pathological conditions. Autophagy represents a complex multistep cellular process, wherein a double membrane vesicle (the autophagosome captures organelles and proteins and delivers them to the lysosome. This natural and destructive mechanism allows the cells to degrade and recycle its cellular components, such as amino acids, monosaccharides, and lipids. Autophagy is an important mechanism used by cells to clear pathogenic organism and deal with stresses. Therefore, it has also been implicated in several diseases, predominantly in cancer. In fact, pharmacological stimulation or inhibition of autophagy have been proposed as approaches to develop new therapeutic treatments of cancers. In conclusion, this blue-print autophagy (so defined because it is induced and/or inhibited by marine natural products represents a new strategy for the future of biomedicine and of biotechnology in cancer treatment.

  4. System-wide Benefits of Intermeal Fasting by Autophagy.

    Science.gov (United States)

    Martinez-Lopez, Nuria; Tarabra, Elena; Toledo, Miriam; Garcia-Macia, Marina; Sahu, Srabani; Coletto, Luisa; Batista-Gonzalez, Ana; Barzilai, Nir; Pessin, Jeffrey E; Schwartz, Gary J; Kersten, Sander; Singh, Rajat

    2017-12-05

    Autophagy failure is associated with metabolic insufficiency. Although caloric restriction (CR) extends healthspan, its adherence in humans is poor. We established an isocaloric twice-a-day (ITAD) feeding model wherein ITAD-fed mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. We hypothesized that ITAD feeding will provide two intervals of intermeal fasting per circadian period and induce autophagy. We show that ITAD feeding modifies circadian autophagy and glucose/lipid metabolism that correlate with feeding-driven changes in circulating insulin. ITAD feeding decreases adiposity and, unlike CR, enhances muscle mass. ITAD feeding drives energy expenditure, lowers lipid levels, suppresses gluconeogenesis, and prevents age/obesity-associated metabolic defects. Using liver-, adipose-, myogenic-, and proopiomelanocortin neuron-specific autophagy-null mice, we mapped the contribution of tissue-specific autophagy to system-wide benefits of ITAD feeding. Our studies suggest that consuming two meals a day without CR could prevent the metabolic syndrome. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana.

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    Soto-Burgos, Junmarie; Bassham, Diane C

    2017-01-01

    Autophagy is a degradation process in which cells break down and recycle their cytoplasmic contents when subjected to environmental stress or during cellular remodeling. The Arabidopsis thaliana SnRK1 complex is a protein kinase that senses changes in energy levels and triggers downstream responses to enable survival. Its mammalian ortholog, AMPK, and yeast ortholog, Snf-1, activate autophagy in response to low energy conditions. We therefore hypothesized that SnRK1 may play a role in the regulation of autophagy in response to nutrient or energy deficiency in Arabidopsis. To test this hypothesis, we determined the effect of overexpression or knockout of the SnRK1 catalytic subunit KIN10 on autophagy activation by abiotic stresses, including nutrient deficiency, salt, osmotic, oxidative, and ER stress. While wild-type plants had low basal autophagy activity in control conditions, KIN10 overexpression lines had increased autophagy under these conditions, indicating activation of autophagy by SnRK1. A kin10 mutant had a basal level of autophagy under control conditions similar to wild-type plants, but activation of autophagy by most abiotic stresses was blocked, indicating that SnRK1 is required for autophagy induction by a wide variety of stress conditions. In mammals, TOR is a negative regulator of autophagy, and AMPK acts to activate autophagy both upstream of TOR, by inhibiting its activity, and in a parallel pathway. Inhibition of Arabidopsis TOR leads to activation of autophagy; inhibition of SnRK1 did not block this activation. Furthermore, an increase in SnRK1 activity was unable to induce autophagy when TOR was also activated. These results demonstrate that SnRK1 acts upstream of TOR in the activation of autophagy in Arabidopsis.

  6. TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana.

    Science.gov (United States)

    Pu, Yunting; Luo, Xinjuan; Bassham, Diane C

    2017-01-01

    Autophagy is a critical process for recycling of cytoplasmic materials during environmental stress, senescence and cellular remodeling. It is upregulated under a wide range of abiotic stress conditions and is important for stress tolerance. Autophagy is repressed by the protein kinase target of rapamycin (TOR), which is activated in response to nutrients and in turn upregulates cell growth and translation and inhibits autophagy. Down-regulation of TOR in Arabidopsis thaliana leads to constitutive autophagy and to decreased growth, but the relationship to stress conditions is unclear. Here, we assess the extent to which TOR controls autophagy activation by abiotic stress. Overexpression of TOR inhibited autophagy activation by nutrient starvation, salt and osmotic stress, indicating that activation of autophagy under these conditions requires down-regulation of TOR activity. In contrast, TOR overexpression had no effect on autophagy induced by oxidative stress or ER stress, suggesting that activation of autophagy by these conditions is independent of TOR function. The plant hormone auxin has been shown previously to up-regulate TOR activity. To confirm the existence of two pathways for activation of autophagy, dependent on the stress conditions, auxin was added exogenously to activate TOR, and the effect on autophagy under different conditions was assessed. Consistent with the effect of TOR overexpression, the addition of the auxin NAA inhibited autophagy during nutrient deficiency, salt and osmotic stress, but not during oxidative or ER stress. NAA treatment was unable to block autophagy induced by a TOR inhibitor or by a mutation in the TOR complex component RAPTOR1B , indicating that auxin is upstream of TOR in the regulation of autophagy. We conclude that repression of auxin-regulated TOR activity is required for autophagy activation in response to a subset of abiotic stress conditions.

  7. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy

    DEFF Research Database (Denmark)

    Farkas, Thomas; Daugaard, Mads; Jaattela, Marja

    2011-01-01

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered...... for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new...

  8. An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

    Science.gov (United States)

    Dagdas, Yasin F; Belhaj, Khaoula; Maqbool, Abbas; Chaparro-Garcia, Angela; Pandey, Pooja; Petre, Benjamin; Tabassum, Nadra; Cruz-Mireles, Neftaly; Hughes, Richard K; Sklenar, Jan; Win, Joe; Menke, Frank; Findlay, Kim; Banfield, Mark J; Kamoun, Sophien; Bozkurt, Tolga O

    2016-01-01

    Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001 PMID:26765567

  9. Roles of autophagy in male reproductive development in plants

    Directory of Open Access Journals (Sweden)

    Shigeru eHanamata

    2014-09-01

    Full Text Available Autophagy, a major catabolic pathway in eukaryotic cells, is essential in development, maintenance of cellular homeostasis, immunity and programmed cell death (PCD in multicellular organisms. In plant cells, autophagy plays roles in recycling of proteins and metabolites including lipids, and is involved in many physiological processes such as abiotic and biotic stress responses. However, its roles during reproductive development had remained poorly understood. Quantitative live cell imaging techniques for the autophagic flux and genetic studies in several plant species have recently revealed significant roles of autophagy in developmental processes, regulation of PCD and lipid metabolism. We here review the novel roles of autophagic fluxes in plant cells, and discuss their possible significance in PCD and metabolic regulation, with particular focus on male reproductive development during the pollen maturation.

  10. Characterization of early autophagy signaling by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

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

    2014-01-01

    . To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon...... revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group...... induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis...

  11. The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

    2018-02-01

    The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

  12. Tuning flux: autophagy as a target of heart disease therapy

    Science.gov (United States)

    Xie, Min; Morales, Cyndi R.; Lavandero, Sergio; Hill, Joseph A.

    2013-01-01

    Purpose of review Despite maximum medical and mechanical support therapy, heart failure remains a relentlessly progressive disorder with substantial morbidity and mortality. Autophagy, an evolutionarily conserved process of cellular cannibalization, has been implicated in virtually all forms of cardiovascular disease. Indeed, its role is context dependent, antagonizing or promoting disease depending on the circumstance. Here, we review current understanding of the role of autophagy in the pathogenesis of heart failure and explore this pathway as a target of therapeutic intervention. Recent findings In preclinical models of heart disease, cardiomyocyte autophagic flux is activated; indeed, its role in disease pathogenesis is the subject of intense investigation to define mechanism. Similarly, in failing human heart of a variety of etiologies, cardiomyocyte autophagic activity is upregulated, and therapy, such as with mechanical support systems, elicits declines in autophagy activity. However, when suppression of autophagy is complete, rapid and catastrophic cell death occurs, consistent with a model in which basal autophagic flux is required for proteostasis. Thus, a narrow zone of ‘optimal’ autophagy seems to exist. The challenge moving forward is to tune the stress-triggered autophagic response within that ‘sweet spot’ range for therapeutic benefit. Summary Whereas we have known for some years of the participation of lysosomal mechanisms in heart disease, it is only recently that upstream mechanisms (autophagy) are being explored. The challenge for the future is to dissect the underlying circuitry and titrate the response into an optimal, proteostasis-promoting range in hopes of mitigating the ever-expanding epidemic of heart failure. PMID:21415729

  13. The cell on the edge of life and death: Crosstalk between autophagy and apoptosis.

    Science.gov (United States)

    Kasprowska-Liśkiewicz, Daniela

    2017-09-21

    Recently, the crosstalk between autophagy and apoptosis has attracted broader attention. Basal autophagy serves to maintain cell homeostasis, while the upregulation of this process is an element of stress response that enables the cell to survive under adverse conditions. Autophagy may also determine the fate of the cell through its interactions with cell death pathways. The protein networks that control the initiation and the execution phase of these two processes are highly interconnected. Several scenarios for the crosstalk between autophagy and apoptosis exist. In most cases, the activation of autophagy represents an attempt of the cell to cope with stress, and protects the cell from apoptosis or delays its initiation. Generally, the simultaneous activation of pro-survival and pro-death pathways is prevented by the mutual inhibitory crosstalk between autophagy and apoptosis. But in some circumstances, autophagy or the proteins of the core autophagic machinery may promote cellular demise through excessive self-digestion (so-called "autophagic cell death") or by stimulating the activation of other cell death pathways. It is controversial whether cells actually die via autophagy, which is why the term "autophagic cell death" has been under intense debate lately. This review summarizes the recent findings on the multilevel crosstalk between autophagy and apoptosis in aspects of common regulators, mutual inhibition of these processes, the stimulation of apoptosis by autophagy or autophagic proteins and finally the role of autophagy as a death-execution mechanism.

  14. Autophagy deficiency in macrophages enhances NLRP3 inflammasome activity and chronic lung disease following silica exposure

    International Nuclear Information System (INIS)

    Jessop, Forrest; Hamilton, Raymond F.; Rhoderick, Joseph F.; Shaw, Pamela K.; Holian, Andrij

    2016-01-01

    Autophagy is an important metabolic mechanism that can promote cellular survival following injury. The specific contribution of autophagy to silica-induced inflammation and disease is not known. The objective of these studies was to determine the effects of silica exposure on the autophagic pathway in macrophages, as well as the general contribution of autophagy in macrophages to inflammation and disease. Silica exposure enhanced autophagic activity in vitro in Bone Marrow derived Macrophages and in vivo in Alveolar Macrophages isolated from silica-exposed mice. Impairment of autophagy in myeloid cells in vivo using Atg5 fl/fl LysM-Cre + mice resulted in enhanced cytotoxicity and inflammation after silica exposure compared to littermate controls, including elevated IL-18 and the alarmin HMGB1 in the whole lavage fluid. Autophagy deficiency caused some spontaneous inflammation and disease. Greater silica-induced acute inflammation in Atg5 fl/fl LysM-Cre + mice correlated with increased fibrosis and chronic lung disease. These studies demonstrate a critical role for autophagy in suppressing silica-induced cytotoxicity and inflammation in disease development. Furthermore, this data highlights the importance of basal autophagy in macrophages and other myeloid cells in maintaining lung homeostasis. - Highlights: • Silica exposure increases autophagy in macrophages. • Autophagy deficient mice have enhanced inflammation and silicosis. • Autophagy deficiency in macrophages results in greater silica-induced cytotoxicity. • Autophagy deficiency in macrophages increases extracellular IL-18 and HMGB1.

  15. The potential regulatory roles of NAD(+) and its metabolism in autophagy.