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Cellular and Molecular Life Sciences :... Oct 2022Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined...
Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.
Topics: Animals; Humans; Astrocytes; Wortmannin; Encephalitis Viruses, Tick-Borne; Autophagy; Sirolimus; Virus Replication
PubMed: 36283999
DOI: 10.1007/s00018-022-04578-7 -
Frontiers in Medicine 2023We previously identified growth arrest and DNA-damage-inducible gene 34 (GADD34) as a marker of ischemic stroke. In the present study, serum levels of anti-GADD34...
We previously identified growth arrest and DNA-damage-inducible gene 34 (GADD34) as a marker of ischemic stroke. In the present study, serum levels of anti-GADD34 antibodies were found to be significantly higher in patients with acute ischemic stroke or chronic kidney disease compared to healthy donors. We then examined the biological function of GADD34 by transfection into U2OS human osteosarcoma and U87 human glioblastoma cells. Knockdown of GADD34 by siRNA resulted in enhanced cell proliferation, which was reversed by co-knockdown of MDM2. Luciferase reporter assays revealed that the transactivation ability of p53 enhanced by genotoxic anticancer drugs such as camptothecin and etoposide was further potentiated by enforced expression of GADD34 but attenuated by co-transfection with p53 shRNA expression plasmids. Western blotting demonstrated increased p53 protein levels after treatment with camptothecin, which was also potentiated by GADD34 but suppressed by GADD34 siRNA, ATM siRNA, and ATM inhibitor wortmannin. GADD34 levels also increased in response to treatment with camptothecin or adriamycin, and this increase was attenuated by MDM2 siRNA. Immunoprecipitation with anti-GADD34 antibody followed by Western blotting with anti-MDM2 antibodies indicated ubiquitination of GADD34 is mediated by MDM2. Accordingly, GADD34 may function as a ubiquitination decoy to reduce p53 ubiquitination and increase p53 protein levels. Increased neuronal cell death due to activation of p53 by GADD34 may account for the elevated serum levels of anti-GADD34 antibodies observed in patients with acute ischemic stroke.
PubMed: 37228401
DOI: 10.3389/fmed.2023.1128921 -
Heliyon Sep 2021Delayed healing of diabetic foot ulcers (DFUs) is one of the major consequences of angiopathy caused by hyperglycemia stemming from insulin resistance. Interventions...
Delayed healing of diabetic foot ulcers (DFUs) is one of the major consequences of angiopathy caused by hyperglycemia stemming from insulin resistance. Interventions that improve blood supply and hyperglycemia are essential for treating DFUs. Low-frequency vibration (LFV) promotes peripheral blood flow and wound healing in DFUs, regardless of hyperglycemia. We hypothesized that LFV promotes non-insulin-mediated glucose uptake, which is also referred to as AMPK-mediated glucose uptake, in adipocytes at wound sites, thereby alleviating hyperglycemia, which, in turn, accelerates wound healing. The objective of this study was to identify LFVs that optimally promote glucose uptake in adipocytes and investigate the mechanism underlying enhanced glucose uptake caused by LFV. 3T3-L1 adipocytes were used in this study. LFV was applied at 50 Hz for 40 min/d to investigate the most effective vibration intensity (0-2000 mVpp) and duration (0-7 d) of glucose uptake. We comparatively assessed 2-deoxyglucose (2-DG) uptake in control and vibration groups. To elucidated the mechanism underlying 2-DG uptake induced by LFV, wortmannin and compound C were used to inhibit insulin-mediated GLUT4 translocation and AMPK activation, respectively. Additionally, GLUT4 translocation to the plasma membrane was assessed using immunofluorescence image analysis. Our results indicated that 2-DG uptake in the 1000 and 1500 mVpp groups was higher than that in the control group (p = 0.0372 and 0.0018, respectively). At 1000 mVpp, 2-DG uptake in the 5- and 7-d groups was higher than that in the non-vibration group (p = 0.0169 and 0.0452, respectively). Although wortmannin did not inhibit 2-DG uptake, compound C did. GLUT4 translocation to the plasma membrane was not observed in the vibration group adipocytes treated with compound C. Thus, our results indicated that an LFV of 50 Hz, 1000 mVpp, 40 min/d, over 5 d was optimal for accelerating AMPK-mediated GLUT4 translocation and glucose uptake in adipocytes.
PubMed: 34568592
DOI: 10.1016/j.heliyon.2021.e07897 -
Cardiovascular Therapeutics 2022Remote ischemic conditioning (RIC) is a cardioprotective method in ischemia/reperfusion (I/R) injury. This study investigated the mechanism of Rho-kinase-mediated...
OBJECTIVE
Remote ischemic conditioning (RIC) is a cardioprotective method in ischemia/reperfusion (I/R) injury. This study investigated the mechanism of Rho-kinase-mediated autophagy in RIC.
METHODS
Sixty male Sprague-Dawley rats were randomly divided into six groups: sham, I/R, RIC, I/R+fasudil, RIC+wortmannin, and RIC+fasudil+wortmannin. Throughout the experiment, mean arterial pressure and heart rate were continuously monitored. Histopathology and ultrastructure and myocardial enzymes' expression were evaluated to determine the degree of cardiac injury. The protein expression of the Rho-kinase substrates myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1), autophagy-related protein light chain 3-II (LC3-II) and Beclin 1, and protein kinase B (AKT) was measured in the myocardial tissue.
RESULTS
Compared with the sham group, the mean arterial pressure and heart rate were decreased, myocardial enzyme levels were increased, and myocardial damage was aggravated in the I/R group; however, RIC improved these alterations. The expression of phosphorylated MLC and MYPT1 was lower, while LC3-II, Beclin 1, and phospho-AKT expression levels were higher in the RIC group compared with the I/R group. Obviously, treatment of the I/R group rats with fasudil, a Rho-kinase inhibitor, significantly ameliorated the I/R effects, whereas treatment of the RIC group rats with wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor, inhibited the RIC protective effects. Moreover, the rats in the RIC+fasudil+wortmannin group showed similar changes to those in the RIC+wortmannin group.
CONCLUSION
These results showed that RIC protected the myocardium from I/R injury by suppressing Rho-kinase and the underlying mechanism may be related to enhancing autophagy via the PI3K/AKT pathway.
Topics: Animals; Autophagy; Ischemia; Male; Myocardial Reperfusion Injury; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; rho-Associated Kinases
PubMed: 35082919
DOI: 10.1155/2022/6806427 -
Frontiers in Immunology 2018Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic...
Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.
Topics: Adenine; Adenosine Triphosphate; Autophagy; Autophagy-Related Protein 5; Cell Line; Humans; Inflammation Mediators; Interleukin-1beta; Lipopolysaccharides; Macrolides; Microtubule-Associated Proteins; Neutrophils; Protein Transport; RNA, Small Interfering; Secretory Pathway; Serine Proteases; Wortmannin
PubMed: 29515581
DOI: 10.3389/fimmu.2018.00269 -
Scientific Reports Sep 2017The interplay between inflammation and lymphangiogenesis is mediated by various cytokines. However, most of these molecules and their associated mechanism are yet to be...
The interplay between inflammation and lymphangiogenesis is mediated by various cytokines. However, most of these molecules and their associated mechanism are yet to be defined. Here, we explored the role of IL-33 in modulating inflammation-induced lymphangiogenesis (ILA) and its underlying mechanisms using an ILA mouse model and a lymphatic endothelial cell (LEC) line. Our results show that IL-33 promoted the proliferation, migration and tube formation of LECs and ILA in vivo. The pro-lymphangiogenic activity of IL-33 was abolished by ST2 blockage. In mechanisms, IL-33 induced the phosphorylation of Akt/eNOS to produce NO in LECs. The IL-33-induced Akt/eNOS activation was suppressed by the PI3K-specific-inhibitor wortmannin, and NO-production was inhibited by both wortmannin and the NO synthase-inhibitor NMA. Knock-down of ST2 or TRAF6 suppressed Akt/eNOS phosphorylation and NO production. The reduction of NO treated with wortmannin or NMA abolished the promoting effects of IL-33 on the chemotactic motility and tube formation of HDLECs. In vivo, IL-33-induced ILA was also impaired in eNOS mice. In conclusion, our study is the first to show that IL-33 promotes inflammation-induced lymphangiogenesis via a ST2/TRAF6-mediated Akt/eNOS/NO signalling pathway. This findings may provide us more opportunities to treat inflammation and lymphangiogenesis associated diseases.
Topics: Animals; Biomarkers; Cell Movement; Cell Proliferation; Cornea; Disease Models, Animal; Endothelial Cells; Immunohistochemistry; Inflammation; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Lymphangiogenesis; Male; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type III; Proto-Oncogene Proteins c-akt; Signal Transduction; TNF Receptor-Associated Factor 6
PubMed: 28878285
DOI: 10.1038/s41598-017-10894-x -
Aging Aug 2021Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with a poor prognosis and a high recurrence rate. gene is frequently mutated in...
Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with a poor prognosis and a high recurrence rate. gene is frequently mutated in breast cancer, with as the hotspot mutation reported in TNBC. We used the ZINC database to screen natural compounds that could be structurally modified to develop drugs targeting the PIK3CA H1047R mutant protein in the PI3K pathway. The LibDock module showed that 2,749 compounds could strongly bind to the PIK3CA H1047R protein. Ultimately, the top 20 natural ligands with high LibDock scores were used for further analyses including assessment of ADME (absorption, distribution, metabolism, and excretion), toxicity, stability, and binding affinity. ZINC000004098448 and ZINC000014715656 were selected as the safest drug candidates with strong binding affinity to PIK3CA H1047R, no hepatotoxicity, less carcinogenicity, better plasma protein binding (PPB) properties, and enhanced intestinal permeability and absorption than the two reference drugs, PKI-402 and wortmannin. Moreover, their lower potential energies than those of PIK3CA H1047R confirmed the stability of the ligand-receptor complex under physiological conditions. ZINC000004098448 and ZINC000014715656 are thus safe and stable leads for designing drugs against PIK3CA H1047R as part of a targeted therapeutic approach for patients with TNBC.
Topics: Binding Sites; Class I Phosphatidylinositol 3-Kinases; Computational Biology; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Ligands; Models, Molecular; Mutation, Missense; Phenylurea Compounds; Pyrimidines; Signal Transduction; Triple Negative Breast Neoplasms; Wortmannin
PubMed: 34415239
DOI: 10.18632/aging.203409 -
Journal of Controlled Release :... Apr 2018Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States; the predominant cause for mortality is metastasis to distant organs (e.g.,...
Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States; the predominant cause for mortality is metastasis to distant organs (e.g., lung). A major problem limiting the success of chemotherapy in metastatic CRC is the inability to target tumor tissues selectively and avoid severe side effects to normal tissues and organs. Here, we demonstrate polymeric nanoparticles (PNPs) entrapping chemotherapeutic agents provide a new therapeutic option for treating CRC that has metastasized to the lung. PNPs assembled from FDA approved biocompatible block copolymer accumulated predominantly in lung tissue. PNPs showed negligible accumulation in liver, spleen and kidneys, which was confirmed by fluorescent nanoparticle imaging and analysis of PI3K inhibition in the organs. PNPs entrapping PI3K inhibitors (i.e., wortmannin and PX866) suppressed CRC lung metastasis growth, and SN-38-loaded PNPs completely eliminated CRC lung metastasis. Our results demonstrate that polymer-drug nanoparticles offer a new approach to reduce toxicity of cancer therapy and has the potential to improve outcomes for patients with lung metastasis.
Topics: Animals; Colorectal Neoplasms; Drug Carriers; Gonanes; HT29 Cells; Humans; Irinotecan; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Nanoparticles; Phosphoinositide-3 Kinase Inhibitors; Polymers; Topoisomerase I Inhibitors; Wortmannin
PubMed: 29421609
DOI: 10.1016/j.jconrel.2018.02.008 -
Cell Chemical Biology Jul 2017To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early...
To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages.
Topics: Action Potentials; Aminopyridines; Androstadienes; Bridged Bicyclo Compounds, Heterocyclic; CD11b Antigen; Endosomes; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Lung; Lysosomal-Associated Membrane Protein 1; Lysosomes; Macrophages; Macrophages, Peritoneal; Patch-Clamp Techniques; Thiazolidines; Transient Receptor Potential Channels; Wortmannin; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; rab7 GTP-Binding Proteins
PubMed: 28732201
DOI: 10.1016/j.chembiol.2017.05.025 -
Frontiers in Physiology 2021Glioblastomas (GBs) are among the most common tumors with high malignancy and invasiveness of the central nervous system. Several alterations in protein kinase and ion...
Glioblastomas (GBs) are among the most common tumors with high malignancy and invasiveness of the central nervous system. Several alterations in protein kinase and ion channel activity are involved to maintain the malignancy. Among them, phosphatidylinositol 3-kinase (PI3K) activity and intermediate conductance calcium-activated potassium (KCa3.1) current are involved in several aspects of GB biology. By using the electrophysiological approach and noise analysis, we observed that KCa3.1 channel activity is LY294002-sensitive and Wortmannin-resistant in accordance with the involvement of PI3K class IIβ (PI3KC2β). This modulation was observed also during the endogenous activation of KCa3.1 current with histamine. The principal action of PI3KC2β regulation was the reduction of open probability in intracellular free calcium saturating concentration. An explanation based on the "three-gate" model of the KCa3.1 channel by PI3KC2β was proposed. Based on the roles of KCa3.1 and PI3KC2β in GB biology, a therapeutic implication was suggested to prevent chemo- and radioresistance mechanisms.
PubMed: 35069252
DOI: 10.3389/fphys.2021.790922