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Redox Biology Jun 2023Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE)...
Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (HS). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of HS replacement by using the mitochondrial-targeted HS donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the HS donor. Experiments performed in the aorta of CSE mice confirmed that reduced levels of HS not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves HS/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the HS/NO interplay in the vasoactive response.
Topics: Mice; Animals; Cattle; Hydrogen Sulfide; Nitric Oxide; Endothelial Cells; Phosphatidylinositol 3-Kinases; Hyperglycemia; Nitric Oxide Synthase Type III; Acetylcholine
PubMed: 36913800
DOI: 10.1016/j.redox.2023.102657 -
International Journal of Molecular... Nov 2020Mesoporous bioactive glass nanospheres (NanoMBGs) have high potential for clinical applications. However, the impact of these nanoparticles on the immune system needs to...
Mesoporous bioactive glass nanospheres (NanoMBGs) have high potential for clinical applications. However, the impact of these nanoparticles on the immune system needs to be addressed. In this study, the biocompatibility of SiO-CaO NanoMBGs was evaluated on different mouse immune cells, including spleen cells subsets, bone marrow-derived dendritic cells (BMDCs), or cell lines like SR.D10 Th2 CD4 lymphocytes and DC2.4 dendritic cells. Flow cytometry and confocal microscopy show that the nanoparticles were rapidly and efficiently taken up in vitro by T and B lymphocytes or by specialized antigen-presenting cells (APCs) like dendritic cells (DCs). Nanoparticles were not cytotoxic and had no effect on cell viability or proliferation under T-cell (anti-CD3) or B cell (LPS) stimuli. Besides, NanoMBGs did not affect the balance of spleen cell subsets, or the production of intracellular or secreted pro- and anti-inflammatory cytokines (TNF-α, IFN-γ, IL-2, IL-6, IL-10) by activated T, B, and dendritic cells (DC), as determined by flow cytometry and ELISA. T cell activation surface markers (CD25, CD69 and Induced Costimulator, ICOS) were not altered by NanoMBGs. Maturation of BMDCs or DC2.4 cells in vitro was not altered by NanoMBGs, as shown by expression of Major Histocompatibility Complex (MHC) and costimulatory molecules (CD40, CD80, CD86), or IL-6 secretion. The effect of wortmannin and chlorpromazine indicate a role for phosphoinositide 3-kinase (PI3K), actin and clathrin-dependent pathways in NanoMBG internalization. We thus demonstrate that these NanoMBGs are both non-toxic and non-inflammagenic for murine lymphoid cells and myeloid DCs despite their efficient intake by the cells.
Topics: Animals; Bone Marrow Cells; Calcium Compounds; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Cytokines; Dendritic Cells; Female; Immunologic Techniques; Inflammation Mediators; Lymphocyte Activation; Male; Materials Testing; Mice; Mice, Inbred C57BL; Nanospheres; Oxides; Porosity; Silicon Dioxide; Spleen
PubMed: 33167415
DOI: 10.3390/ijms21218291 -
Bioscience Reports Oct 2020The present study was to determine the roles of Angiotensin (Ang) II in the growth of lymphoma in nude mice and the proliferation and viability of the human Natural...
The present study was to determine the roles of Angiotensin (Ang) II in the growth of lymphoma in nude mice and the proliferation and viability of the human Natural Killer/T (NK/T)-cell lymphoma cell line SNK-6, and the activation of downstream signaling pathway. Lymphoma samples and corresponding normal tissues were obtained from lymphoma patients. Proliferation of SNK-6 cells was detected by CCK8 or MTT assay. The levels of Ang II and its receptor Ang II type 1 receptor (AT1R) were higher in lymphoma tissues than those in control tissues. Ang II increased the lymphoma volume and size in nude mice, the proliferation and viability and the proliferating cell nuclear antigen (PCNA) and Ki67 levels of SNK-6 cells. Losartan, an antagonist of AT1R, reduced lymphoma volume and size in nude mice, and the proliferation and viability and the PCNA and Ki67 levels of SNK-6 cells. The levels of phosphorylated phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-Akt) were increased by Ang II and then reduced by losartan in SNK-6 cells. The proliferation and viability of SNK-6 cells were increased by Ang II, but these increases were inhibited by PI3K inhibitor wortmannin and Akt inhibitor MK2206. The increases of PCNA and Ki67 induced by Ang II were inhibited by wortmannin or MK2206 in SNK-6 cells. These results indicate that Ang II/AT1R is activated in lymphoma, and Ang II promotes the progression of lymphoma in nude mice and the proliferation and viability of SNK-6 cells via activating PI3K/Akt signaling pathway.
Topics: Aged; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Heterocyclic Compounds, 3-Ring; Humans; Losartan; Lymph Nodes; Lymphoma, Extranodal NK-T-Cell; Male; Middle Aged; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, Angiotensin, Type 1; Signal Transduction; Wortmannin; Xenograft Model Antitumor Assays
PubMed: 32969473
DOI: 10.1042/BSR20202388 -
Neural Regeneration Research Jul 2021We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents. Retrograde axonal...
We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents. Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3'-kinase inhibitor, wortmannin, via the sciatic nerve results in loss of ipsilateral lumbar motor neurons proportional to the level of drug administered. Motor neuron loss was detected by choline acetyltransferase (ChAT) immunostaining and with a transgenic thy1-eGFP marker. The short half-life of wortmannin generates minimal wound spread, and wortmannin does not affect axon transport, as determined by co-injection of a pseudorabies virus tracer. Using quantitative transcript analysis, we found that ChAT transcripts significantly decreased at 14 days post-delivery of 1 μg wortmannin, relative to sham controls, and remained low after 90 days. Smaller effects were observed with 200 ng and 100 ng wortmannin. Wortmannin also generated a transient and significant increase in astrocyte Gfap transcripts after 14 days with a return to control levels at 90 days. Treated mice had hind limb spasticity and a forced motor function defect that was quantified using a water exit test. Controls rapidly exit a shallow water tray, and wortmannin treated animals were up to 12-fold slower, a phenotype that persisted for at least 3 months. Thus the focal delivery of wortmannin to motor neurons generates a reproducible and scalable injury that can facilitate quantitative studies on neural regeneration and repair. The efficacy of sciatic nerve suicide transport can also explain neurotoxin-mediated selective loss of motor neurons in diseases such as amyotrophic lateral sclerosis. All procedures were performed at Rutgers under established Institutional Animal Care and Use protocols (eIACUC_TR201800022, approved on March 20, 2018).
PubMed: 33318406
DOI: 10.4103/1673-5374.301032 -
Journal of Cellular and Molecular... May 2022Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1α (HIF-1α) and glucose...
Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1α (HIF-1α) and glucose transporter 1 (Glut-1) double knockout reduced tumour biological behaviour in laryngeal carcinoma cells. However, their radioresistance mechanism remains unclear. In this study, cell viability was determined by CCK8 assay. Glucose uptake capability was evaluated by measurement of F-fluorodeoxyglucose radioactivity. A tumour xenograft model was established by subcutaneous injection of Tu212 cells. Tumour histopathology was determined by haematoxylin and eosin staining, immunohistochemical staining, and TUNEL assays. Signalling transduction was evaluated by Western blotting. We found that hypoxia induced radioresistance in Tu212 cells accompanied by increased glucose uptake capability and activation of the PI3K/Akt/mTOR pathway. Inhibition of PI3K/Akt/mTOR activity abolished hypoxia-induced radioresistance and glucose absorption. Mechanistic analysis revealed that hypoxia promoted higher expressions of HIF-1α and Glut-1. Moreover, the PI3K/Akt/mTOR pathway was a positive mediator of HIF-1α and/or Glut-1 in the presence of irradiation. HIF-1α and/or Glut-1 knockout significantly reduced cell viability, glucose uptake and PI3K/Akt/mTOR activity, all of which were induced by hypoxia in the presence of irradiation. In vivo analysis showed that knockout of HIF-1α and/or Glut-1 also inhibited tumour growth by promoting cell apoptosis, more robustly compared with the PI3K inhibitor wortmannin, particularly in tumours with knockout of both HIF-1α and Glut-1. HIF-1α and/or Glut-1 knockout also abrogated PI3K/Akt/mTOR signalling transduction in tumour tissues, in a manner similar to wortmannin. HIF-1α and/or Glut-1 knockout facilitated radiosensitivity in laryngeal carcinoma Tu212 cells by regulation of the PI3K/Akt/mTOR pathway.
Topics: Animals; CRISPR-Cas Systems; Carcinoma; Cell Line, Tumor; Glucose; Glucose Transporter Type 1; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Laryngeal Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Radiation Tolerance; TOR Serine-Threonine Kinases; Wortmannin
PubMed: 35415942
DOI: 10.1111/jcmm.17303 -
Biomedicine & Pharmacotherapy =... Jan 2024Bladder cancer cells possess unique adaptive capabilities: shaped by their environment, cells face a complex chemical mixture of metabolites and xenobiotics accompanied...
Bladder cancer cells possess unique adaptive capabilities: shaped by their environment, cells face a complex chemical mixture of metabolites and xenobiotics accompanied by physiological mechanical cues. These responses might translate into resistance to chemotherapeutical regimens and can largely rely on autophagy. Considering molecules capable of rewiring tumor plasticity, compounds of natural origin promise to offer valuable options. Fungal derived metabolites, such as bafilomycin and wortmannin are widely acknowledged as autophagy inhibitors. Here, their potential to tune bladder cancer cells´ adaptability to chemical and physical stimuli was assessed. Additionally, dietary occurring mycotoxins were also investigated, namely deoxynivalenol (DON, 0.1-10 µM) and fusaric acid (FA, 0.1-1 mM). Endowing a Janus' face behavior, DON and FA are on the one side described as toxins with detrimental health effects. Concomitantly, they are also explored experimentally for selective pharmacological applications including anticancer activities. In non-cytotoxic concentrations, bafilomycin (BAFI, 1-10 nM) and wortmannin (WORT, 1 µM) modified cell morphology and reduced cancer cell migration. Application of shear stress and inhibition of mechano-gated PIEZO channels reduced cellular sensitivity to BAFI treatment (1 nM). Similarly, for FA (0.5 mM) PIEZO1 expression and inhibition largely aligned with the modulatory potential on cancer cells motility. Additionally, this study highlighted that the activity profile of compounds with similar cytotoxic potential (e.g. co-incubation DON with BAFI or FA with WORT) can diverge substantially in the regulation of cell mechanotransduction. Considering the interdependence between tumor progression and response to mechanical cues, these data promise to provide a novel viewpoint for the study of chemoresistance and associated pathways.
Topics: Humans; Mechanotransduction, Cellular; Wortmannin; Autophagy; Antineoplastic Agents; Urinary Bladder Neoplasms; Ion Channels
PubMed: 38042111
DOI: 10.1016/j.biopha.2023.115942 -
BMJ Open Gastroenterology Jul 2023Stellate cells are responsible for liver and pancreas fibrosis and strictly correlate with tumourigenesis. Although their activation is reversible, an exacerbated...
OBJECTIVE
Stellate cells are responsible for liver and pancreas fibrosis and strictly correlate with tumourigenesis. Although their activation is reversible, an exacerbated signalling triggers chronic fibrosis. Toll-like receptors (TLRs) modulate stellate cells transition. TLR5 transduces the signal deriving by the binding to bacterial flagellin from invading mobile bacteria.
DESIGN
Human hepatic and pancreatic stellate cells were activated by the administration of transforming growth factor-beta (TGF-β). TLR5 was transiently knocked down by short-interference RNA transfection. Reverse Transcription-quantitativePCR and western blot were performed to analyse the transcript and protein level of TLR5 and the transition players. Fluorescence microscopy was performed to identify these targets in spheroids and in the sections of murine fibrotic liver.
RESULTS
TGF-β-activated human hepatic and pancreatic stellate cells showed an increase of expression. knockdown blocked the activation of those stellate cells. Furthermore, TLR5 busted during murine liver fibrosis and co-localised with the inducible Collagen I. Flagellin suppressed , and expression after the administration of TGF-β. Instead, the antagonist of TLR5 did not block the effect of TGF-β. Wortmannin, a specific AKT inhibitor, induced but not and transcript and protein level.
CONCLUSION
TGF-β-mediated activation of hepatic and pancreatic stellate cells requires the over-expression of TLR5. Instead, its autonomous signalling inhibits the activation of the stellate cells, thus prompting a signalling through different regulatory pathways.
Topics: Animals; Humans; Mice; Flagellin; Liver Cirrhosis; Pancreatic Stellate Cells; Toll-Like Receptor 5
PubMed: 37433685
DOI: 10.1136/bmjgast-2023-001148 -
Computers in Biology and Medicine Jul 2022The 2019 novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a major challenge threatening the...
BACKGROUND
The 2019 novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a major challenge threatening the global healthcare system. Respiratory virus infection is the most common cause of asthma attacks, and thus COVID-19 may contribute to an increase in asthma exacerbations. However, the mechanisms of COVID-19/asthma comorbidity remain unclear.
METHODS
The "Limma" package or "DESeq2" package was used to screen differentially expressed genes (DEGs). Alveolar lavage fluid datasets of COVID-19 and asthma were obtained from the GEO and GSV database. A series of analyses of common host factors for COVID-19 and asthma were conducted, including PPI network construction, module analysis, enrichment analysis, inference of the upstream pathway activity of host factors, tissue-specific analysis and drug candidate prediction. Finally, the key host factors were verified in the GSE152418 and GSE164805 datasets.
RESULTS
192 overlapping host factors were obtained by analyzing the intersection of asthma and COVID-19. FN1, UBA52, EEF1A1, ITGB1, XPO1, NPM1, EGR1, EIF4E, SRSF1, CCR5, PXN, IRF8 and DDX5 as host factors were tightly connected in the PPI network. Module analysis identified five modules with different biological functions and pathways. According to the degree values ranking in the PPI network, EEF1A1, EGR1, UBA52, DDX5 and IRF8 were considered as the key cohost factors for COVID-19 and asthma. The HO, VEGF, IL-1 and Wnt signaling pathways had the strongest activities in the upstream pathways. Tissue-specific enrichment analysis revealed the different expression levels of the five critical host factors. LY294002, wortmannin, PD98059 and heparin might have great potential to evolve into therapeutic drugs for COVID-19 and asthma comorbidity. Finally, the validation dataset confirmed that the expression of five key host factors were statistically significant among COVID-19 groups with different severity and healthy control subjects.
CONCLUSIONS
This study constructed a network of common host factors between asthma and COVID-19 and predicted several drugs with therapeutic potential. Therefore, this study is likely to provide a reference for the management and treatment for COVID-19/asthma comorbidity.
Topics: Asthma; Bronchoalveolar Lavage Fluid; COVID-19; Computational Biology; DEAD-box RNA Helicases; Gene Expression Profiling; Humans; Hydrogen Peroxide; Interferon Regulatory Factors; Protein Interaction Maps; SARS-CoV-2; Serine-Arginine Splicing Factors
PubMed: 35751199
DOI: 10.1016/j.compbiomed.2022.105601 -
Cancers Jul 2020In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs,...
In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs, miRs) miR526b/miR655 in poorly metastatic breast cancer cell lines promotes aggressive cancer phenotypes in vitro and in vivo. Additionally, miR526b/miR655 expression is significantly higher in human breast tumors, and high miR526b/miR655 expression is associated with poor prognosis. However, the roles of miR526b/miR655 in hypoxia are unknown. To test the relationship between miR526b/miR655 and hypoxia, we used various in vitro, in silico, and in situ assays. In normoxia, miRNA-high aggressive breast cancer cell lines show higher HIF-1α expression than miRNA-low poorly metastatic breast cancer cell lines. To test direct involvement of miR526b/miR655 in hypoxia, we analyzed miRNA-high cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) compared to controls (MCF7 and SKBR3). CoCl-induced hypoxia in breast cancer further promotes mRNA and protein expression while reducing expression (a negative HIF-1α regulator), especially in miRNA-high cell lines. Hypoxia enhances oxidative stress, epithelial to mesenchymal transition, cell migration, and vascular mimicry more prominently in MCF7-miR526b/MCF7-miR655 cell lines compared to MCF7 cells. Hypoxia promotes inflammatory and angiogenesis marker (, , , ) expression in all miRNA-high cells. Hypoxia upregulates miR526b/miR655 expression in MCF7 cells, thus observed enhancement of hypoxia-induced functions in MCF7 could be attributed to miR526b/miR655 upregulation. In silico bioinformatics analysis shows miR526b/miR655 regulate (a negative regulator of ) and (positive regulator of and ) expression by downregulation of transcription factors , , and . Hypoxia-enhanced functions in miRNA-high cells are inhibited by COX-2 inhibitor (Celecoxib), EP4 antagonist (ONO-AE3-208), and irreversible PI3K/Akt inhibitor (Wortmannin). This establishes that hypoxia enhances miRNA functions following the COX-2/EP4/PI3K/Akt pathways and this pathway can serve as a therapeutic target to abrogate hypoxia and miRNA induced functions in breast cancer. In situ, expression is significantly higher in human breast tumors ( = 96) compared to non-cancerous control tissues ( = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, expression was significantly higher in ER-positive, PR-positive, and HER2-negative breast tumors. Data extracted from the TCGA database also show a strong correlation between and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia.
PubMed: 32707933
DOI: 10.3390/cancers12082008 -
PloS One 2022Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment...
Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment when these organisms are challenged with draught, high salinity or metabolic stress (e.g. acetate accumulation). In turn, morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying Saccharomyces cerevisiae and its vacuole as models, conserved molecular mechanisms responsible for fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to metabolism or osmoregulation remain elusive. Herein we describe a new fluorometric assay to measure yeast vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a metabolic stressor, triggers vacuole fission and show it blocks homotypic vacuole fusion in vitro. Similarly, hypertonic stress induced by sorbitol or glucose caused robust vacuole fission in vitro whilst inhibiting fusion. Using wortmannin to inhibit phosphatidylinositol (PI) -kinases or rGyp1-46 to inactivate Rab-GTPases, we show that acetate stress likely targets PI signaling, whereas osmotic stress affects Rab signaling on vacuole membranes to stimulate fission. This study sets the stage for further investigation into the mechanisms that change vacuole morphology to support cell metabolism and osmoregulation.
Topics: Acetates; Membrane Fusion; Osmotic Pressure; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 35834522
DOI: 10.1371/journal.pone.0271199