scholarly journals Creating a bottleneck: Robust LC3B lipidation analysis by adjusting autophagic flux with low concentrations of Bafilomycin A1

2021 ◽  
Author(s):  
Martina P Liebl ◽  
Sarah C Meister ◽  
Lisa Frey ◽  
Kristina Hendrich ◽  
Anja Klemmer ◽  
...  
Keyword(s):  
Late Stage ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Bafilomycin A1 ◽  
Low Concentrations ◽  
Flux Quantification ◽  
The Creation ◽  
Flux Increase

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

scholarly journals Robust LC3B lipidation analysis by precisely adjusting autophagic flux

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Martina P. Liebl ◽  
Sarah C. Meister ◽  
Lisa Frey ◽  
Kristina Hendrich ◽  
Anja Klemmer ◽  
...  
Keyword(s):  
Late Stage ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Bafilomycin A1 ◽  
Flux Quantification ◽  
The Creation ◽  
Flux Increase

AbstractAutophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

scholarly journals Effect of early-stage autophagy inhibition in BRAFV600E autophagy-dependent brain tumor cells

2019 ◽  
Vol 10 (9) ◽  
Author(s):  
Shadi Zahedi ◽  
Brent E. Fitzwalter ◽  
Andrew Morin ◽  
Sydney Grob ◽  
Michele Desmarais ◽  
...  
Keyword(s):  
Cell Survival ◽  
Tumor Cells ◽  
Late Stage ◽  
Early Stage ◽  
Specific Effect ◽  
Cns Tumors ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Tumor Cell Death ◽  
Autophagy Inhibition

Abstract Autophagy is a multistage process. Progress within the field has led to the development of agents targeting both early (initiation) and late (fusion) stages of this process. The specific stage of autophagy targeted may influence cancer treatment outcomes. We have previously shown that central nervous system (CNS) tumors with the BRAFV600E mutation are autophagy dependent, and late-stage autophagy inhibition improves the response to targeted BRAF inhibitors (BRAFi) in sensitive and resistant cells. Drugs directed toward initiation of autophagy have been shown to reduce tumor cell death in some cancers, but have not been assessed in CNS tumors. We investigated early-stage inhibition for autophagy-dependent CNS tumors. BRAFi-sensitive and resistant AM38 and MAF794 cell lines were evaluated for the response to pharmacologic and genetic inhibition of ULK1 and VPS34, two crucial subunits of the autophagy initiation complexes. Changes in autophagy were monitored by western blot and flow cytometry. Survival was evaluated in short- and long-term growth assays. Tumor cells exhibited a reduced autophagic flux with pharmacologic and genetic inhibition of ULK1 or VPS34. Pharmacologic inhibition reduced cell survival in a dose-dependent manner for both targets. Genetic inhibition reduced cell survival and confirmed that it was an autophagy-specific effect. Pharmacologic and genetic inhibition were also synergistic with BRAFi, irrespective of RAFi sensitivity. Inhibition of ULK1 and VPS34 are potentially viable clinical targets in autophagy-dependent CNS tumors. Further evaluation is needed to determine if early-stage autophagy inhibition is equal to late-stage inhibition to determine the optimal clinical target for patients.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Gadolinium Oxide Nanoparticles Enhance the Cytotoxicity of Chemotherapeutic Drugs by Blocking Autophagic Flux in Human Ovarian Cancer Cells

2020 ◽  
Vol 16 ◽  
Author(s):  
Zhixiong Xie ◽  
Tianyu Zhang ◽  
Cheng Zhong
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Hela Cells ◽  
Late Stage ◽  
Ovarian Cancer Cells ◽  
Oxide Nanoparticles ◽  
Autophagic Flux ◽  
Human Ovarian Cancer ◽  
Chemotherapeutic Drugs ◽  
Chemotherapy Drugs

Background: During chemotherapy, drugs can damage cancer cells’ DNA and cytomembrane structure, and then induce cell death. However, autophagy can increase the chemotherapy resistance of cancer cells, reducing the effect of chemotherapy. Objective: To block the autophagic flux in cancer cells, it is vital to enhance the anti-cancer efficacy of chemotherapy drugs; for this purpose, we test the gadolinium oxide nanoparticles (Gd2O3 NPs)’ effect on autophagy. Methods: The cytotoxicity of Gd2O3 NPs on HeLa cells was evaluated by a (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then, monodasylcadaverine staining, immunofluorescence, immunoblot and apoptosis assay were conducted to evaluate the effect of Gd2O3 NPs on autophagy and efficacy of chemotherapy drugs in human ovarian cancer cells. Results: We found that Gd2O3 NPs, which have great potential for use as a contrast agent in magnetic resonance imaging, could block the late stage of autophagic flux in a dose-dependent manner and then cause autophagosome accumulation in HeLa cells. When co-treated with 8 μg/mL Gd2O3 NPs and 5 μg/mL cisplatin, the number of dead HeLa cells increased by about 20% compared with cisplatin alone. We observed the same phenomenon in cisplatin-resistant COC1/DDP cells. Conclusion: We conclude that Gd2O3 NPs can block the late stage of autophagic flux and enhance the cytotoxicity of chemotherapeutic drugs in human ovarian cancer cells. Thus, the nanoparticles have significant potential for use in both diagnosis and therapy of solid tumor.

scholarly journals Surgical procedures suppress autophagic flux in the kidney

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Carolyn N. Brown ◽  
Daniel Atwood ◽  
Deepak Pokhrel ◽  
Sara J. Holditch ◽  
Christopher Altmann ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Mammalian Target Of Rapamycin ◽  
Kidney Injury ◽  
Compensatory Hypertrophy ◽  
Autophagic Flux ◽  
Bafilomycin A1 ◽  
Concomitant Increase ◽  
Sham Surgery ◽  
And Function ◽  
Pro Inflammatory Cytokines

AbstractMany surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.

Effects of ethanol on gastric epithelial cell phospholipid dynamics and cellular function

1987 ◽  
Vol 252 (2) ◽  
pp. G237-G243
Author(s):  
R. E. Bailey ◽  
R. A. Levine ◽  
J. Nandi ◽  
E. H. Schwartzel ◽  
D. H. Beach ◽  
...  
Keyword(s):  
Membrane Structure ◽  
Lipid Membrane ◽  
Proton Nuclear Magnetic Resonance ◽  
Resonance Spectroscopy ◽  
Dependent Manner ◽  
Chloride Uptake ◽  
Peak Intensity ◽  
Low Concentrations ◽  
Resonance Band ◽  
Dose Dependent

The lipid profile of isolated gastric superficial epithelial cells (SEC) was evaluated by proton nuclear magnetic resonance spectroscopy (1H-NMR). The most conspicuous resonance band in SEC spectra was due to the protons of +N(CH3)3 groups of phosphatidylcholine and, to a lesser degree, other phospholipid derivatives, on the basis of their chemical shift and addition of purified phospholipids. NMR of cell lysates and phospholipid extracts of SEC in deutero-chloroform provided further spectral resolution of these components. Phospholipase or ethanol treatments of SEC produced membrane disorganization reflected as increased peak intensity of the phospholipid signals. In addition, ethanol, in a dose-dependent manner, attenuated paranitrophenyl phosphatase activity, which correlated with inhibition of total and ouabain-sensitive 86Rubidium chloride uptake by SEC. This study suggests that NMR used in conjunction with other biochemical techniques can monitor SEC membrane structure-function relationships. NMR is a potentially powerful noninvasive probe to show changes in lipid membrane organization induced by low concentrations of ethanol (1%) and may indicate an early sign of "cytotoxicity" in intact SEC.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

scholarly journals (Pro)renin receptor regulates autophagy and apoptosis in podocytes exposed to high glucose

2015 ◽  
Vol 309 (3) ◽  
pp. E302-E310 ◽  
Author(s):  
Caixia Li ◽  
Helmy M. Siragy
Keyword(s):  
Signaling Pathway ◽  
High Glucose ◽  
Caspase 3 ◽  
Mtor Signaling ◽  
Autophagic Flux ◽  
Bafilomycin A1 ◽  
Mtor Signaling Pathway ◽  
Autophagosome Formation ◽  
Podocyte Injury ◽  
Protein Levels

High glucose reduces autophagy and enhances apoptosis of podocytes. Previously, we reported that high glucose induced podocyte injury through upregulation of the (pro)renin receptor (PRR). We hypothesized that increasing PRR reduces autophagy and increases apoptosis of mouse podocytes exposed to high glucose via activation of the PI3K/Akt/mTOR signaling pathway. Mouse podocytes were cultured in normal (5 mmol/l) or high (25 mmol/l) d-glucose for 48 h. High glucose significantly increased mRNA and protein levels of PRR, phosphorylation of PI3K/Akt/mTOR, and p62. In contrast, high glucose decreased activation of UNC-51-like kinase-1 (ULK1) by phosphorylating Ser757 and protein levels of microtubule-associated protein-1 light chain 3B (LC3B)-II and Lamp-2. Bafilomycin A1 increased LC3BII and p62 accumulation in high-glucose-treated cells. High glucose reduced the autophagic flux. Confocal microscopy studies showed significant reduction in the protein level of LC3B in response to high glucose. Cyto-ID autophagy staining showed a significant decrease in autophagosome formation with high glucose. In the absence of PRR, activation of Akt with sc-79 or mTOR with MHY-1485 increased p62 accumulation. Caspase-3/7 activity and apoptosis monitored by TUNEL assay were significantly increased in podocytes treated with high glucose. PRR siRNA significantly reversed the effects of high glucose. Based on these data, we conclude that high glucose decreases autophagy and increases apoptosis in mouse podocytes through the PRR/PI3K/Akt/mTOR signaling pathway.

LukS-PV Induced HOXA9 Degradation in Acute Myeloid Leukemia Cells via Autophagy

2021 ◽  
Author(s):  
Ping Qiang ◽  
Chao Fang ◽  
Kaidi Song ◽  
Lan Shi ◽  
Yuanyuan Dai ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Prominent Feature ◽  
Concentration Dependent Manner ◽  
Over Expression ◽  
Autophagy Induction ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Aberrant over-expression of HOXA9 is a prominent feature of AML driven by multiple oncogenes, thus therapeutic degradation of HOXA9 by autophagy may be an effective treatment strategy for AML. PVL is a pore forming cytotoxin secreted by Staphylococcus aureus, and it is composed of two subunits - LukS-PV and LukF-PV. Here, we show that LukS-PV can stimulate the conversion of LC3-I to LC3-II in AML cells in a concentration-dependent manner, and autophagic vacuoles can be found in LukS-PV-treated THP-1 cells. Furthermore, we find that an accumulation of LC3-positive structures in AML cells exposed to LukS-PV, indicating that an increased autophagic flux were formed. Therefore, LukS-PV induced autophagy of AML cells. We also demonstrated that LukS-PV could regulate the expression of HOXA9 at the protein level. HOXA9 molecules were detected in autophagosomes after LukS-PV treatment, indicating that autophagy induction accounted for the degradation of HOXA9.

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