scholarly journals STF-62247 accumulates in lysosomes and blocks late stages of autophagy to selectively target von Hippel-Lindau-inactivated cells

2019 ◽  
Vol 316 (5) ◽  
pp. C605-C620 ◽  
Author(s):  
Nadia Bouhamdani ◽  
Dominique Comeau ◽  
Kevin Cormier ◽  
Sandra Turcotte
Keyword(s):  
Cellular Localization ◽  
Time Dependent ◽  
Mechanistic Study ◽  
Autophagic Flux ◽  
Cancer Type ◽  
Selective Cytotoxicity ◽  
Homeostatic Process ◽  
Von Hippel Lindau ◽  
Small Compound ◽  
Late Stages

Autophagy is a highly conserved, homeostatic process by which cytosolic components reach lysosomes for degradation. The roles played by different autophagic processes in cancer are complex and remain cancer type and stage dependent. Renal cell carcinoma (RCC) is the most common subtype of kidney cancer and is characterized by the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Our previous study identified a small compound, STF-62247, as an autophagy-modulating molecule causing selective cytotoxicity for VHL-inactivated cells. This present study investigates the effects of STF-62247 specifically on the macroautophagic flux to better characterize its mechanism of action in RCC. Our results clearly demonstrate that this compound is a potent blocker of late stages of autophagy. We show that inhibiting autophagy by CRISPR knockouts of autophagy-related genes rendered VHL-deficient cells insensitive to STF-62247, uncovering the importance of the autophagic pathway in STF-selective cell death. By exploiting the autofluorescence of STF-62247, we pinpointed its cellular localization to lysosomes. Finally, in response to prolonged STF treatments, we show that VHL-proficient cells are able to surmount the block in late stages of autophagy by restoring their lysosome numbers. Conversely, an increase in autophagic vesicles accompanied by a time-dependent decrease in lysosomes was observed in VHL-deficient cells. This is the first mechanistic study investigating STF-62447’s effects on the autophagic flux in RCC. Importantly, our study reclassifies STF-62247 as a blocker of later stages of autophagy and highlights the possibility of blocking this process through lysosome disruption in VHL-mutated RCCs.

scholarly journals STF-62247 blocks late stages of autophagy by disrupting lysosomal physiology

2018 ◽  
Author(s):  
Nadia Bouhamdani ◽  
Dominique Comeau ◽  
Kevin Cormier ◽  
Sandra Turcotte
Keyword(s):  
Cathepsin D ◽  
Potential Role ◽  
Suppressor Gene ◽  
Mechanistic Study ◽  
Autophagic Flux ◽  
Von Hippel Lindau ◽  
Lysosomal Ph ◽  
Trafficking Defects ◽  
Late Stages ◽  
Acidic Compartments

ABSTRACTSTF-62247 was previously identified as a promising compound able to selectively target the loss of the tumor suppressor gene von Hippel-Lindau (VHL) in renal cell carcinomas. This present work investigates the effect of STF-62247 on the autophagic flux. Our investigations show that STF-62247 blocks late stages of autophagy through lysosomal disruption. Indeed, STF-62247 localizes at lysosomes and causes unregulated swelling of these acidic compartments in VHL-mutated cells, linking a potential role for VHL in lysosomal integrity. Knock-outs of BECN1 and ATG5 were able to rescue the viability of VHL-mutated cells in response to STF-62247 but did not rescue the lysosomal swelling. In fact, neutralizing the lysosomal pH by inhibiting the vacuolar H+-ATPase completely rescued this phenotype. Moreover, we show that STF-62247 disrupts endocytic routes and causes cathepsin D trafficking defects. This mechanistic study is the first to characterize STF-62447 as a novel lysosomotropic compound. Importantly, our study re-classifies STF-62247 as a blocker of later stages of autophagy and highlights its potential usage as a powerful new tool for endocytic and autophagy-related research.

scholarly journals Her2-Targeted Therapy Induces Autophagy in Esophageal Adenocarcinoma Cells

2018 ◽  
Vol 19 (10) ◽  
pp. 3069 ◽  
Author(s):  
Félice Janser ◽  
Olivia Adams ◽  
Vanessa Bütler ◽  
Anna Schläfli ◽  
Bastian Dislich ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Esophageal Adenocarcinoma ◽  
Acquired Resistance ◽  
Therapy Resistance ◽  
Her2 Status ◽  
Autophagic Flux ◽  
Cancer Type ◽  
Cancer Resistance ◽  
Her2 Positive ◽  
Autophagy Inhibition

Esophageal adenocarcinoma (EAC) is a highly lethal cancer type with an overall poor survival rate. Twenty to thirty percent of EAC overexpress the human epidermal growth factor receptor 2 (Her2), a transmembrane receptor tyrosine kinase promoting cell growth and proliferation. Patients with Her2 overexpressing breast and gastroesophageal cancer may benefit from Her2 inhibitors. Therapy resistance, however, is well documented. Since autophagy, a lysosome-dependent catabolic process, is implicated in cancer resistance mechanisms, we tested whether autophagy modulation influences Her2 inhibitor sensitivity in EAC. Her2-positive OE19 EAC cells showed an induction in autophagic flux upon treatment with the small molecule Her2 inhibitor Lapatinib. Newly generated Lapatinib-resistant OE19 (OE19 LR) cells showed increased basal autophagic flux compared to parental OE19 (OE19 P) cells. Based on these results, we tested if combining Lapatinib with autophagy inhibitors might be beneficial. OE19 P showed significantly reduced cell viability upon double treatment, while OE19 LR were already sensitive to autophagy inhibition alone. Additionally, Her2 status and autophagy marker expression (LC3B and p62) were investigated in a treatment-naïve EAC patient cohort (n = 112) using immunohistochemistry. Here, no significant correlation between Her2 status and expression of LC3B and p62 was found. Our data show that resistance to Her2-directed therapy is associated with a higher basal autophagy level, which is not per se associated with Her2 status. Therefore, we propose that autophagy may contribute to acquired resistance to Her2-targeted therapy in EAC, and that combining Her2 and autophagy inhibition might be beneficial for EAC patients.

scholarly journals A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

2020 ◽  
Vol 11 ◽  
Author(s):  
Chun-Hsien Wu ◽  
Chin Heng Gan ◽  
Lan-Hui Li ◽  
Jen-Che Chang ◽  
Shin-Tai Chen ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Membrane Integrity ◽  
Mechanistic Study ◽  
Autophagic Flux ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Inflammatory Agent ◽  
Nlrp3 Inflammasome Activation ◽  
Autophagy Induction ◽  
Anti Inflammatory

Conjugated polyenes are a class of widely occurring natural products with various biological functions. We previously identified 4-hydroxy auxarconjugatin B (4-HAB) as anti‐inflammatory agent with an IC50 of ~20 µM. In this study, we synthesized a new anti‐inflammatory 4-HAB analogue, F240B, which has an IC50 of less than 1 µM. F240B dose-dependently induced autophagy by increasing autophagic flux, LC3 speck formation and acidic vesicular organelle formation. F240B inhibited NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome activation through autophagy induction. In a mechanistic study, F240B inhibited interleukin (IL)-1β (IL-1β) precursor expression, promoted degradation of NLRP3 and IL-1β, and reduced mitochondrial membrane integrity loss in an autophagy-dependent manner. Additionally, F240B inhibited apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and speck formation without affecting the interaction between NLRP3 and ASC or NIMA-related kinase 7 (NEK7) and double-stranded RNA-dependent kinase (PKR). Furthermore, F240B exerted in vivo anti-inflammatory activity by reducing the intraperitoneal influx of neutrophils and the levels of IL-1β, active caspase-1, IL-6 and monocyte chemoattractant protein-1 (MCP-1) in lavage fluids in a mouse model of uric acid crystal-induced peritonitis. In conclusion, F240B attenuated the NLRP3 inflammasome through autophagy induction and can be developed as an anti-inflammatory agent in the future.

scholarly journals Alterations of Plasmin Activity, Plasminogen Levels and Activity of Anti-Plasmins During Endotoxin Shock in Dogs

1977 ◽  
Author(s):  
A. O. Aasen ◽  
M. J. Gallimore ◽  
K. Ohlsson ◽  
E. Amundsen
Keyword(s):  
Intravenous Infusion ◽  
Lethal Dose ◽  
Endotoxin Shock ◽  
Time Dependent ◽  
Plasmin Activity ◽  
Endotoxin Infusion ◽  
E Coli ◽  
Research Division ◽  
Late Stages

Endotoxin shock was induced in dogs by intravenous infusion of a lethal dose of E. coli endotoxin over a period of 3 hours. Typical changes of cardiovascular parameters were found and evidence of an intravascular clotting process was observed. Spontaneous plasmin activity and “immediate” and “time dependent” antiplasmin activities were determined by means of assays utilizing the chromogenic tripeptide derivative S-2251(Kabi Peptide Research Division, Mölndal, Sweden). Levels of plasminogen, α2-macrolobulin (α2-M) , and ai-antitrypsin(α1-AT) were determined immunochemically. During shock, gradually decreasing values of “immediate” antiplasmin and α2M were observed. During the late stages of shock “immediate” antiplasmin was found to be reduced by up to 89 per cent and α2M up to 50 per cent of pre endotoxin infusion values. A less marked lowering of “time dependent” antiplasmin and α1-AT also occurred during shock. These changes of plasma antiplasmins were accompanied by decreasing values of plasminogen and evidence of plasmin activity. These findings indicate that plasminogen is converted to plasmin during endotoxin shock and emphasize the role of antiplasmins in the pathophysiology of endotoxin shock.

scholarly journals P2X7R-mediated Autophagic Impairment Contributes to Central Sensitization in a chronic Migraine Model with Recurrent Nitroglycerin Stimulation in Mice

2020 ◽  
Author(s):  
Li Jiang ◽  
Yixin Zhang ◽  
Feng Jing ◽  
Ting Long ◽  
Guangcheng Qin ◽  
...  
Keyword(s):  
Chronic Migraine ◽  
Central Sensitization ◽  
Cellular Localization ◽  
Microglial Activation ◽  
Purinergic Receptor ◽  
Radiant Heat ◽  
Underlying Mechanism ◽  
Autophagic Flux ◽  
Pathophysiological Mechanism

Abstract Background: Central sensitization is an important pathophysiological mechanism of chronic migraine (CM). According to our previous studies, microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to the central sensitization. The P2X7 receptor (P2X7R) is a purinergic receptor expressed in microglia and participates in central sensitization in chronic pain, but its role in CM is unclear. Numerous studies have shown that P2X7R regulates the level of autophagy and that autophagy affects the microglial activation and inflammation. Recently, autophagy has been shown to be involved in neuropathic pain, but there is no information about autophagy in CM. Therefore, the current study investigated the role of P2X7R in CM and its underlying mechanism, focusing on autophagy regulation.Methods: The CM model was established by repeated intraperitoneal injection of nitroglycerin (NTG) in mice. A Von Frey filament and radiant heat were used to assess the mechanical and thermal hypersensitivity. Western blotting and immunofluorescence assays were performed to detect the expression of P2X7R, autophagy-related proteins, and the cellular localization of P2X7R. To determine the role of P2X7R and autophagy in CM, we detected the effects of the autophagy inducer, rapamycin (RAPA) and P2X7R antagonist, Brilliant Blue G (BBG), on pain behavior and the expression of calcitonin gene-related peptide (CGRP) and c-fos. In addition, the effect of RAPA and BBG on microglial activation and subsequent inflammation were investigated.Results: The expression of P2X7R was increased and was mainly colocalized with microglia in the TNC following recurrent NTG administration. The autophagic flux was blocked in CM, which was characterized by up-regulated LC3-II, and accumulated autophagy substrate protein, p62. RAPA significantly improved the basal rather than acute hyperalgesia. BBG alleviated both basal and acute hyperalgesia. BBG activated the level of autophagic flux. RAPA and BBG inhibited the activation of microglia, limited the inflammatory response, and reduced the expression of CGRP and c-fos. Conclusions: Our results demonstrate the dysfunction of the autophagic process in CM. Activated autophagy may have a preventive effect on migraine chronification. P2X7R contributes to central sensitization through mediating autophagy regulation and might become a potential target for CM.

scholarly journals The interrupted effect of autophagic flux and lysosomal function induced by graphene oxide in p62-dependent apoptosis of F98 cells

2020 ◽  
Author(s):  
Chao Zhang ◽  
Xiaoli Feng ◽  
Longwen He ◽  
Yaqing Zhang ◽  
Longquan Shao
Keyword(s):  
Graphene Oxide ◽  
Annexin V ◽  
Time Dependent ◽  
Immunofluorescence Staining ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Western Blots ◽  
Lysosomal Function ◽  
Abnormal Accumulation ◽  
Induced Apoptosis

Abstract Abstract Background : Graphene oxide (GO) nanoparticles (NPs) have been widely applied in various fields, especially in biomedical applications. Extensive studies have suggested that GO can pass through the blood-brain barrier (BBB) and induce abnormal autophagy and cytotoxicity in the central nervous system (CNS). However, the effect and specific mechanism of GO on astrocytes, the most abundant cells in the brain still has not been extensively investigated. Results: In this study, we systematically explored the toxicity and mechanism of GO exposure in the rat astroglioma-derived F98 cell line using molecular biological techniques (immunofluorescence staining, flow cytometry and Western blot) at the subcellular level and the signaling pathway level. Cells exposed to GO exhibited decreased cell viability and increased lactate dehydrogenase (LDH) release in a concentration- and time-dependent manner. GO-induced autophagy was evidenced by transmission electron microscopy (TEM) and immunofluorescence staining. Western blots showed that LC3II/I and p62 were upregulated and PI3K/Akt/mTOR was downregulated. Detection of lysosomal acidity and cathepsin B activity assay indicated the impairment of lysosomal function. Annexin V-FITC-PI detection showed the occurrence of apoptosis after GO exposure. The decrease in mitochondrial membrane potential (MMP) with an accompanying upregulation of cleaved caspase-3 and Bax/Bcl-2 further suggested that endogenous signaling pathways were involved in GO-induced apoptosis. Conclusion: The exposure of F98 cells to GO can elicit concentration- and time-dependent toxicological effects. Additionally, increased autophagic response can be triggered after GO treatment and that the blocking of autophagy flux plays a vital role in GO cytotoxicity, which was determined to be related to dysfunction of lysosomal degradation. Importantly, the abnormal accumulation of autophagic substrate p62 protein can induce capase-3-mediated apoptosis. Inhibition of abnormal accumulation of autophagic cargo could alleviate the occurrence of GO-induced apoptosis in F98 cells. Keyword: Graphene oxide; Astrocyte; p62; Autophagy; Apoptosis

scholarly journals Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2315
Author(s):  
Marius W. Baeken ◽  
Katja Weckmann ◽  
Philip Diefenthäler ◽  
Jan Schulte ◽  
Kamran Yusifli ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Human Fibroblasts ◽  
Sirtuin 1 ◽  
Autophagic Flux ◽  
Molecular Function ◽  
Knock Down ◽  
Human Disorders ◽  
Total Protein Expression ◽  
Protein Lipidation ◽  
Starvation Conditions

Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.

scholarly journals Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1673
Author(s):  
Lilya Lehka ◽  
Małgorzata Topolewska ◽  
Dominika Wojton ◽  
Olena Karatsai ◽  
Paloma Alvarez-Suarez ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Cellular Localization ◽  
Fold Increase ◽  
Time Dependent ◽  
Myoblast Differentiation ◽  
Focal Contact ◽  
Myosin Vi ◽  
Cytoskeleton Organization ◽  
Hindlimb Muscles ◽  
Primary Myoblast

We have previously postulated that unconventional myosin VI (MVI) could be involved in myoblast differentiation. Here, we addressed the mechanism(s) of its involvement using primary myoblast culture derived from the hindlimb muscles of Snell’s waltzer mice, the natural MVI knockouts (MVI-KO). We observed that MVI-KO myotubes were formed faster than control heterozygous myoblasts (MVI-WT), with a three-fold increase in the number of myosac-like myotubes with centrally positioned nuclei. There were also changes in the levels of the myogenic transcription factors Pax7, MyoD and myogenin. This was accompanied by changes in the actin cytoskeleton and adhesive structure organization. We observed significant decreases in the levels of proteins involved in focal contact formation, such as talin and focal adhesion kinase (FAK). Interestingly, the levels of proteins involved in intercellular communication, M-cadherin and drebrin, were also affected. Furthermore, time-dependent alterations in the levels of the key proteins for myoblast membrane fusion, myomaker and myomerger, without effect on their cellular localization, were observed. Our data indicate that in the absence of MVI, the mechanisms controlling cytoskeleton organization, as well as myoblast adhesion and fusion, are dysregulated, leading to the formation of aberrant myotubes.

scholarly journals The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review

2020 ◽  
Vol 21 (23) ◽  
pp. 8933
Author(s):  
Cédric H. G. Neutel ◽  
Jhana O. Hendrickx ◽  
Wim Martinet ◽  
Guido R. Y. De Meyer ◽  
Pieter-Jan Guns
Keyword(s):  
Systematic Review ◽  
English Language ◽  
Treatment Strategies ◽  
Cochrane Library ◽  
Autophagic Flux ◽  
Protective Effects ◽  
Eligibility Criteria ◽  
Lysosomal Function ◽  
Homeostatic Process ◽  
Valvular Calcification

Background: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN). Objective: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy. Data sources: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015–2020 (92%). Conclusion: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.

Fibroblast fate regulation by time dependent TGF-β1 and IL-10 stimulation in biomimetic 3D matrices

2017 ◽  
Vol 5 (9) ◽  
pp. 1858-1867 ◽  
Author(s):  
Jiranuwat Sapudom ◽  
Xiancheng Wu ◽  
Marina Chkolnikov ◽  
Michael Ansorge ◽  
Ulf Anderegg ◽  
...  
Keyword(s):  
Wound Healing ◽  
Time Dependent ◽  
Myofibroblast Differentiation ◽  
Late Stages ◽  
Tgf Β1

Myofibroblast differentiation and de-differentiation at early and late stages of wound healing is modelled in 3D biomimetic matrices based on collagen and fibronectin in vitro.

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