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BMC Biology Jan 2024Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative....
BACKGROUND
Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells.
RESULTS
Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death.
CONCLUSIONS
Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.
Topics: Humans; Autophagosomes; Glioblastoma; Autophagy; Proteomics; Lysosomes; Endoplasmic Reticulum Stress
PubMed: 38287397
DOI: 10.1186/s12915-024-01829-w -
Frontiers in Immunology 2023In blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established model with which to study...
In blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established model with which to study macrophage function and behaviour. However, the use of plasmatocytes as a macrophage model has been limited by a historical perspective that plasmatocytes represent a homogenous population of cells, in contrast to the high levels of heterogeneity of vertebrate macrophages. Recently, a number of groups have reported transcriptomic approaches which suggest the existence of plasmatocyte heterogeneity, while we identified enhancer elements that identify subpopulations of plasmatocytes which exhibit potentially pro-inflammatory behaviours, suggesting conservation of plasmatocyte heterogeneity in . These plasmatocyte subpopulations exhibit enhanced responses to wounds and decreased rates of efferocytosis when compared to the overall plasmatocyte population. Interestingly, increasing the phagocytic requirement placed upon plasmatocytes is sufficient to decrease the size of these plasmatocyte subpopulations in the embryo. However, the mechanistic basis for this response was unclear. Here, we examine how plasmatocyte subpopulations are modulated by apoptotic cell clearance (efferocytosis) demands and associated signalling pathways. We show that loss of the phosphatidylserine receptor Simu prevents an increased phagocytic burden from modulating specific subpopulation cells, while blocking other apoptotic cell receptors revealed no such rescue. This suggests that Simu-dependent efferocytosis is specifically involved in determining fate of particular subpopulations. Supportive of our original finding, mutations in (the homolog of ), a calcium-permeable channel which operates downstream of Simu, phenocopy mutants. Furthermore, we show that Amo is involved in the acidification of the apoptotic cell-containing phagosomes, suggesting that this reduction in pH may be associated with macrophage reprogramming. Additionally, our results also identify Ecdysone receptor signalling, a pathway related to control of cell death during developmental transitions, as a controller of plasmatocyte subpopulation identity. Overall, these results identify fundamental pathways involved in the specification of plasmatocyte subpopulations and so further validate plasmatocytes as a heterogeneous population of macrophage-like cells within this important developmental and immune model.
Topics: Animals; Drosophila; Drosophila melanogaster; Efferocytosis; Macrophages; Drosophila Proteins
PubMed: 38283366
DOI: 10.3389/fimmu.2023.1310117 -
Open Research Europe 2023Cigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease....
INTRODUCTION
Cigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease. However, there remains a knowledge gap in how constituents of cigarette smoke affect vision and retinal biology. We used zebrafish to assess effects of short-term acute exposure to cigarette smoke extract (CSE) on visual behaviour and retinal biology.
METHODS
Zebrafish larvae with a developed visual system at three days post-fertilization (dpf) were exposed to CSE for 4, 24 or 48 hours. Visual behaviour, hyaloid vasculature morphology, retinal histology, oxidative stress gene expression and outer segment phagocytosis were investigated using visual behavioural optokinetic and visual motor response assays (OKR and VMR), microscopy (light, fluorescence and transmission electron microscopy), and real-time PCR.
RESULTS
In zebrafish larvae, 48 hours of CSE treatment resulted in significantly reduced visual behaviour. Larvae treated with 10, 15 or 20 μg/mL CSE showed an average of 13.7, 10.7 or 9.4 saccades per minute, respectively, significantly lower compared with 0.05% DMSO controls (p=0.0093, p=0.0004 and p<0.0001, respectively) that exhibited 19.7 saccades per minute. The diameter of intraocular vessels increased from 4.833 μm in 0.05% DMSO controls to 5.885 μm in the 20 μg/mL CSE-treated larvae (p=0.0333). Biometry analysis highlighted a significant axial length elongation in 20 μg/mL CSE-treated larvae (216.9 μm, p<0.0001) compared to 0.05% dimethyl sulfoxide (DMSO) controls (205.1 μm). Larvae exposed to 20 μg/mL CSE had significantly (p=0.0002) higher numbers of RPE phagosomes compared to vehicle controls (0.1425 and 0.093 phagosomes/μm RPE, respectively).
CONCLUSIONS
Zebrafish larvae with a developed visual system display apparent defects in visual behaviour and retinal biology after acute exposure to CSE, establishing a valuable model to investigate ocular disorders related to cigarette smoke.
PubMed: 38283058
DOI: 10.12688/openreseurope.15491.2 -
International Journal of Molecular... Jan 2024Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by lipid accumulation within the liver. The pathogenesis underlying its development is poorly...
Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by lipid accumulation within the liver. The pathogenesis underlying its development is poorly understood. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and a group 1 carcinogen. The aryl hydrocarbon receptor activation by B[a]P induces cytochrome P450 (CYP) enzymes, contributing to hepatic lipid accumulation. However, the molecular mechanism through which the B[a]P-mediated induction of CYP enzymes causes hepatic lipid accumulation is unknown. This research was conducted to elucidate the role of CYP1B1 in regulating B[a]P-induced lipid accumulation within hepatocytes. B[a]P increased hepatic lipid accumulation, which was mitigated by CYP1B1 knockdown. An increase in the mammalian target of rapamycin (mTOR) by B[a]P was specifically reduced by CYP1B1 knockdown. The reduction of mTOR increased the expression of autophagic flux-related genes and promoted phagolysosome formation. Both the expression and translocation of TFE3, a central regulator of lipophagy, were induced, along with the expression of lipophagy-related genes. Conversely, enhanced mTOR activity reduced TFE3 expression and translocation, which reduced the expression of lipophagy-related genes, diminished phagolysosome production, and increased lipid accumulation. Our results indicate that B[a]P-induced hepatic lipid accumulation is caused by CYP1B1-induced mTOR and the reduction of lipophagy, thereby introducing novel targets and mechanisms to provide insights for understanding B[a]P-induced MASLD.
Topics: Benzo(a)pyrene; Cytochrome P-450 CYP1B1; Liver; Cytochrome P-450 Enzyme System; TOR Serine-Threonine Kinases; Receptors, Aryl Hydrocarbon; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Lipids; Cytochrome P-450 CYP1A1
PubMed: 38279324
DOI: 10.3390/ijms25021324 -
VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.Molecular Biology of the Cell Mar 2024Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma...
Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.
Topics: Receptors, IgG; Phagocytosis; Macrophages; Phagosomes; SNARE Proteins
PubMed: 38265888
DOI: 10.1091/mbc.E23-04-0149 -
Molecular Cancer Jan 2024Eukaryotic cells engage in autophagy, an internal process of self-degradation through lysosomes. Autophagy can be classified as selective or non-selective depending on... (Review)
Review
Eukaryotic cells engage in autophagy, an internal process of self-degradation through lysosomes. Autophagy can be classified as selective or non-selective depending on the way it chooses to degrade substrates. During the process of selective autophagy, damaged and/or redundant organelles like mitochondria, peroxisomes, ribosomes, endoplasmic reticulum (ER), lysosomes, nuclei, proteasomes, and lipid droplets are selectively recycled. Specific cargo is delivered to autophagosomes by specific receptors, isolated and engulfed. Selective autophagy dysfunction is closely linked with cancers, neurodegenerative diseases, metabolic disorders, heart failure, etc. Through reviewing latest research, this review summarized molecular markers and important signaling pathways for selective autophagy, and its significant role in cancers. Moreover, we conducted a comprehensive analysis of small-molecule compounds targeting selective autophagy for their potential application in anti-tumor therapy, elucidating the underlying mechanisms involved. This review aims to supply important scientific references and development directions for the biological mechanisms and drug discovery of anti-tumor targeting selective autophagy in the future.
Topics: Humans; Autophagy; Neoplasms; Autophagosomes; Cell Nucleus; Drug Discovery
PubMed: 38262996
DOI: 10.1186/s12943-024-01934-y -
BioRxiv : the Preprint Server For... Jan 2024Urban particulate matter (uPM) poses significant health risks, particularly to the respiratory system. Fine particles, such as PM, can penetrate deep into the lungs and...
Urban particulate matter (uPM) poses significant health risks, particularly to the respiratory system. Fine particles, such as PM, can penetrate deep into the lungs and exacerbate a range of health problems, including emphysema, asthma, and lung cancer. PM exposure is also linked to extra-pulmonary disorders like heart and neurodegenerative diseases. Moreover, prolonged exposure to elevated PM levels can reduce overall life expectancy. Senescence is a dysfunctional cell state typically associated with age but can also be precipitated by environmental stressors. This study aimed to determine whether uPM could drive senescence in macrophages, an essential cell type involved in particulate phagocytosis-mediated clearance. While it is known that uPM exposure impairs immune function, this deficit is multi-faceted and incompletely understood, partly due to the use of particulates such as diesel exhaust particle (DEP) as a surrogate for true uPM. uPM was collected from several locations in the USA, including Baltimore, Houston, and Phoenix. Bone marrow-derived macrophages (BMDMs) were stimulated with uPM or reference particulates ( DEP) to assess senescence-related parameters. We report that uPM-exposed BMDMs adopt a senescent phenotype characterized by increased IL-1α secretion, senescence-associated β-galactosidase activity, and diminished proliferation. Exposure to allergens failed to elicit such a response, supporting a distinction between different types of environmental exposures. uPM-induced senescence was independent of key macrophage activation pathways, specifically inflammasome and scavenger receptor. However, inhibition of the phagolysosome pathway abrogated senescence markers, supporting this phenotype's attribution to uPM phagocytosis. These data suggest uPM exposure leads to macrophage senescence, which may contribute to immunopathology.
PubMed: 38260346
DOI: 10.1101/2024.01.04.574228 -
Molecules (Basel, Switzerland) Jan 2024Liver fibrosis is the initial pathological process of many chronic liver diseases. Targeting hepatic stellate cell (HSC) activation is an available strategy for the...
Liver fibrosis is the initial pathological process of many chronic liver diseases. Targeting hepatic stellate cell (HSC) activation is an available strategy for the therapy of liver fibrosis. We aimed to explore the anti-liver fibrosis activity and potential mechanism of phomopsterone B (PB) in human HSCs. The results showed that PB effectively attenuated the proliferation of TGF-β1-stimulated LX-2 cells in a concentration-dependent manner at doses of 1, 2, and 4 μM. Quantitative real-time PCR and Western blot assays displayed that PB significantly reduced the expression levels of α-SMA and collagen I/III. AO/EB and Hoechst33342 staining and flow cytometry assays exhibited that PB promoted the cells' apoptosis. Meanwhile, PB diminished the number of autophagic vesicles and vacuolated structures, and the LC3B fluorescent spots indicated that PB could effectively inhibit the accretion of autophagosomes in LX-2 cells. Moreover, rapamycin and MHY1485 were utilized to further investigate the effect of mTOR in autophagy and apoptosis. The results demonstrated that PB regulated autophagy and apoptosis via the mTOR-dependent pathway in LX-2 cells. In summary, this is the first evidence that PB effectively alleviates liver fibrosis in TGF-β1-stimulated LX-2 cells, and PB may be a promising candidate for the prevention of liver fibrosis.
Topics: Humans; Transforming Growth Factor beta1; Autophagy; Liver Cirrhosis; Autophagosomes; Apoptosis
PubMed: 38257331
DOI: 10.3390/molecules29020417 -
Virus Research Mar 2024Respiratory system diseases caused by respiratory viruses are common and exert tremendous pressure on global healthcare system. In our previous studies, we found that...
Respiratory system diseases caused by respiratory viruses are common and exert tremendous pressure on global healthcare system. In our previous studies, we found that Long non-coding RNA NRAV (Lnc NRAV) and its target molecule Rab5c plays a significant role in respiratory virus infection. However, the mechanism by which Rab5c affects virus replication remains unclear. Rab5c, a protein mainly localized on the cell membranes and in early endosomes and phagosomes, participates in endocytosis mediated by clathrin and regulates the fusion of early endosome, maturation of early phagosomes, and autophagy. Therefore, we inferred that Rab5c impacts virus replication, which might be related to endocytosis or autophagy. We selected RSV (respiratory syncytial virus) as a representative enveloped virus and ADV (Adenovirus) as a representative non-enveloped virus to explore the possible mechanism of RSV and ADV replication promoted by Rab5c in A549 cells and in Rab5c-overexpressing mice. Here, we confirmed that the activated Rab5c promotes RSV and ADV replication and the inactivated Rab5c inhibits their replication. However, Rab5c promoting RSV and ADV replication is not mediated by endocytosis rather by autophagy in respiratory epithelial cells. Our study showed that Rab5c upregulates LC3-Ⅱ (microtubule-associated protein 1 light chain 3 beta) protein expression levels by interacting with Beclin1, a key autophagy molecule, which can induce autophagy and promote replication of ADV and RSV. This study enriches the understanding of the interaction between respiratory viruses and Rab5c, providing new insights for virus prevention and treatment.
Topics: Animals; Mice; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Epithelial Cells; Adenoviridae; Autophagy; Virus Replication
PubMed: 38242290
DOI: 10.1016/j.virusres.2024.199324 -
Phytomedicine : International Journal... Mar 2024Autophagy, a cellular process involving lysosomal self-digestion, plays a crucial role in recycling biomolecules and degrading dysfunctional proteins and damaged...
BACKGROUND
Autophagy, a cellular process involving lysosomal self-digestion, plays a crucial role in recycling biomolecules and degrading dysfunctional proteins and damaged organelles. However, in non-small cell lung cancer (NSCLC), cancer cells can exploit autophagy to survive metabolic stress and develop resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which reduce treatment efficacies. Currently, most studies have found that late-stage autophagy inhibitors can hinder EGFR-TKIs resistance, while research on early-stage autophagy inhibitors is still limited.
PURPOSE
This study investigates the mechanism via which the Xie-Bai-San (XBS) formula enhances NSCLC cell sensitivity to gefitinib, revealing the relationship between XBS-induced cell death and the inhibition of autophagosome formation.
METHODS
Cell viability was assessed using CCK-8 and EdU assays, lentivirus transfection was utilized to generate PC9 cells harboring the PIK3CA E545K mutation (referred to as PC9-M), autophagic flux was monitored using mCherry-GFP-LC3 adenovirus. Protein expression and colocalization were observed through immunofluorescence staining. The interaction between Bcl-2 and Beclin-1 in PC9-GR and PC9-M cells was determined via co-immunoprecipitation (Co-IP) assay, cell apoptosis was assessed by flow cytometry and PI staining, and overall survival analysis of lung adenocarcinoma patients was conducted using the TCGA database. In vivo experiments included a patient-derived xenograft (PDX) model with EGFR and PIK3CA mutations and subcutaneous mice xenografts of NSCLC cell lines (PC9 and PC9-GR). In addition, autophagic vesicles in mouse tumor tissues were observed via transmission electron microscopy analysis.
RESULTS
XBS effectively inhibits the proliferation of gefitinib-resistant NSCLC cells and induces apoptosis both in vitro and in vivo. Mechanistically, XBS suppresses gefitinib-induced autophagic flux by inhibiting autophagy through the upregulation of p-mTOR and Bcl-2 and downregulation of Beclin-1. Additionally, XBS enhances the interaction between Bcl-2 and Beclin-1, and the overexpression of Beclin-1 promotes NSCLC cell proliferation and counteracts XBS-induced cell death, while XBS demonstrates minimal impact on autophagosome-lysosome fusion or lysosome function.
CONCLUSION
This study reveals a novel role for the XBS formula in impeding autophagy initiation and demonstrates its potential as a candidate drug to counteract autophagy-induced treatment resistance in NSCLC.
Topics: Humans; Animals; Mice; Carcinoma, Non-Small-Cell Lung; Gefitinib; Beclin-1; Lung Neoplasms; Antineoplastic Agents; Autophagosomes; ErbB Receptors; Quinazolines; Protein Kinase Inhibitors; Drug Resistance, Neoplasm; Apoptosis; Proto-Oncogene Proteins c-bcl-2; Cell Line, Tumor
PubMed: 38232540
DOI: 10.1016/j.phymed.2024.155351