Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.

mCRP-Induced Focal Adhesion Kinase-Dependent Monocyte Aggregation and M1 Polarization, Which Was Partially Blocked by the C10M Inhibitor.

Monomeric C-reactive protein (mCRP) has recently been implicated in the abnormal vascular activation associated with development of atherosclerosis, but it may act more specifically through mechanisms perpetuating damaged vessel inflammation and subsequent aggregation and internalization of resident macrophages. Whilst the direct effects of mCRP on endothelial cells have been characterized, the interaction with blood monocytes has, to our knowledge, not been fully defined. Here we showed that mCRP caused a strong aggregation of both U937 cell line and primary peripheral blood monocytes (PBMs) obtained from healthy donors. Moreover, this increase in clustering was dependent on focal adhesion kinase (FAK) activation (blocked by a specific inhibitor), as was the concomitant adhesive attachment to the plate, which was suggestive of macrophage differentiation. Confocal microscopy confirmed the increased expression and nuclear localization of p-FAK, and cell surface marker expression associated with M1 macrophage polarization (CD11b, CD14, and CD80, as well as iNOS) in the presence of mCRP. Inclusion of a specific CRP dissociation/mCRP inhibitor (C10M) effectively inhibited PBMs clustering, as well as abrogating p-FAK expression, and partially reduced the expression of markers associated with M1 macrophage differentiation. mCRP also increased the secretion of pro-inflammatory cytokines Interleukin-8 (IL-8) and Interleukin-1ß (IL-1ß), without notably affecting MAP kinase signaling pathways; inclusion of C10M did not perturb or modify these effects. In conclusion, mCRP modulates PBMs through a mechanism that involves FAK and results in cell clustering and adhesion concomitant with changes consistent with M1 phenotypical polarization. C10M has potential therapeutic utility in blocking the primary interaction of mCRP with the cells-for example, by protecting against monocyte accumulation and residence at damaged vessels that may be predisposed to plaque development and atherosclerosis.

Gelatin but not type I collagen promotes bacteria phagocytosis in PMA-treated U937 human lymphoma cells.

PURPOSE: Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. MATERIALS AND METHODS: Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. RESULTS: Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. CONCLUSION: This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Azacitidine-Hesperetin Combination Induces S-phase Cell Cycle Arrest and Apoptosis in Human Leukemia Cells.

BACKGROUND/AIM: Chemotherapy drugs for leukemia, such as 5-azacytidine (Aza), have often various adverse effects. Hesperetin (Hes), a naturally occurring compound, is a potential adjuvant agent for anticancer therapy. This study aimed to investigate the effect of an Aza-Hes combination on acute leukemia cell lines, which elucidates the role of combination treatment in leukemia progression. MATERIALS AND METHODS: HL-60 and U937 cells were treated with Aza and Hes at various concentrations or their combination. Cell proliferation and apoptosis was evaluated using the Cell Counting Kit-8 assay and annexin V/propidium iodide staining, respectively. Cell cycle analysis was conducted using flow cytometry. The expression of apoptosis-related and cell cycle-related proteins in leukemia cells was analyzed through western blotting. The synergistic effect of the Aza and Hes agents was estimated using the Chou-Talalay method. RESULTS: We observed that Aza or Hes monotherapy engendered a dose-dependent reduction in HL-60 and U937 cell viability. However, treatment with the Aza-Hes combination for 24 h synergistically inhibited U937 cell proliferation by inducing both apoptosis and S-phase cell cycle arrest. Furthermore, the Aza-Hes combination down-regulated p-ERK and p-c-Jun N-terminal kinase expression and up-regulated p-p38 expression. CONCLUSION: Overall, our findings indicate that the Aza-Hes combination induces apoptosis and S-phase cell-cycle arrest through the mitogen-activated protein kinase pathway. In conclusion, the Aza-Hes combination is a potential antileukemia treatment.

Cigarette smoke extract induces foam cell formation by impairing machinery involved in lipid droplet degradation.

The formation of foam cells, lipid-loaded macrophages, is the hallmark event of atherosclerosis. Since cigarette smoking is a risk factor for developing atherosclerosis, the current study investigated the effects of cigarette smoke extract (CSE) on different events like expressions of genes involved in lipid influx and efflux, lipophagy, etc., that play vital roles in foam cell formation. The accumulation of lipids after CSE treatment U937 macrophage cells was examined by staining lipids with specific dyes: Oil red O and BODIPY493/503. Results showed an accumulation of lipids in CSE-treated cells, confirming foam cell formation by CSE treatment. To decipher the mechanism, the levels of CD36, an ox-LDL receptor, and ABCA1, an exporter of lipids, were examined in CSE-treated and -untreated U937 cells by real-time PCR and immunofluorescence analysis. Consistent with lipid accumulation, an increased level of CD36 and a reduction in ABCA1 were observed in CSE-treated cells. Moreover, CSE treatment caused inhibition of lipophagy-mediated lipid degradation by blocking lipid droplets (LDs)-lysosome fusion and increasing the lysosomal pH. CSE also impaired mitochondrial lipid oxidation. Thus, the present study demonstrates that CSE treatment affects lipid homeostasis by altering its influx and efflux, lysosomal degradation, and mitochondrial utilization, leading to the formation of lipid-loaded foam cells. Moreover, the current study also showed that the leucine supplement caused a significant reduction of CSE-induced foam cell formation in vitro. Thus, the current study provides insight into CS-induced atherosclerosis and an agent to combat the disease.

Formation of free acetaldehydes derived from lipid peroxidation in U937 monocyte-like cells.

Acetaldehyde can be found in human cells as a byproduct of various metabolic pathways, including oxidative processes such as lipid peroxidation. This secondary product of lipid peroxidation plays a role in various pathological processes, leading to various types of civilization diseases. In this study, the formation of free acetaldehyde induced by oxygen-centred radicals was studied in monocyte-like cell line U937. Exposure of U937 cells to peroxyl/alkoxyl radicals induced by azocompound resulted in the formation of free acetaldehyde. Acetaldehyde is formed by the cleavage of fatty acids, which represents the breakdown of fatty acids into smaller fragments initiated by the cyclization of lipid peroxyl radical and ß-scission of lipid alkoxyl radical. The cleavage of fatty acids alters the integrity of the plasma and nuclear membrane, leading to the loss of cell viability. Understanding the pathological processes of acetaldehyde formation is an active area of research with potential implications for preventing and treating various diseases associated with oxidative stress.

The in vitro cytotoxic effects of natural (fibrous epsomite crystals) and synthetic (Epsom salt) magnesium sulfate.

Exposure to mineral fibers represents an occupational and environmental hazard since particulate inhalation leads to several health disorders. However, few data are available on the effect of fibers with high solubility like natural epsomite, a water-soluble fiber with an inhalable size that allows it to penetrate biological systems, with regard to the respiratory tract. This study evaluated the natural (fibrous epsomite) and synthetic (Epsom salt) magnesium sulfate pathogenicity. Investigations have been performed through morpho-functional and biochemical analyses, in an in vitro cell model that usually grows as monocytes, but that under appropriate conditions differentiates into macrophages. These latter, known as alveolar macrophages, if referred to lungs, represent the first line of defense against harmful inhaled stimuli. Morphological observations reveal that, if Epsom salt induces osmotic stress on cell culture, natural epsomite fibers lead to cellular alterations including thickening of the nuclear envelope and degenerated mitochondria. Moreover, the insoluble fraction (impurities) internalized by cells induces diffuse damage characterized at the highest dosage and exposure time by secondary necrosis or necrotic cell death features. Biochemical analyses confirm this mineral behavior that involves MAPK pathway activation, resulting in many different cellular responses ranging from proliferation control to cell death. Epsom salt leads to MAPK/ERK activation, a marker predictive of overall survival. Unlike, natural epsomite induces upregulation of MAPK/p38 protein involved in the phosphorylation of downstream targets driving necrotic cell death. These findings demonstrate natural epsomite toxicity on U937 cell culture, making the inhalation of these fibers potentially hazardous for human health. RESEARCH HIGHLIGHTS: Natural epsomite and synthetic Epsom salt effects have been evaluated in U937 cell model. Epsom salt induces an osmotic cellular stress. Natural epsomite fibers lead to cellular damage and can be considered potentially dangerous for human health.

Phorbal-12-mysristate-13-acetate-induced inflammation is restored by protectin DX through PPARγ in human promonocytic U937 cells.

AIMS: Protectin DX (PDX), a specialized pro-resolving mediator, is an important pharmaceutical compound with potential antioxidant and inflammation-resolving effects. However, the fundamental mechanism by which PDX's ameliorate chronic inflammatory diseases has not yet been elucidated. This study aims to evaluate the anti-inflammatory properties and PPARγ-mediated mechanisms of PDX in phorbal-12-mysristate-13-acetate (PMA)-stimulated human promonocytic U937 cells. MAIN METHODS: We confirmed the effects of PDX on expressions of pro-inflammatory cytokines, mediators, and CD14 using conventional PCR, RT-qPCR, ELISA, and flow cytometry. Using western blotting, immunofluorescence, and reactive oxygen species (ROS) determination, we observed that PDX regulated PMA-induced signaling cascades. Molecular docking analysis and a cellular thermal shift assay were conducted to verify the interaction between PDX and the proliferator-activated receptor-γ (PPARγ) ligand binding domain. Western blotting was then employed to explore the alterations in PPARγ expression levels and validate PDX as a PPARγ full agonist. KEY FINDINGS: PDX attenuated protein and mRNA expression levels of interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in PMA-treated U937 cells. PDX acts as a PPARγ agonist, exerting a modulating effect on the ROS/JNK/c-Fos signaling pathways. Furthermore, PDX reduced human monocyte differentiation antigen CD14 expression levels. SIGNIFICANCE: PPARγ exhibits pro-resolving effects to regulate the excessive inflammation. These results suggest that PDX demonstrates the resolution of inflammation, indicating the potential for therapeutic targeting of chronic inflammatory diseases.

Mesenchymal Stem Cell-Macrophage Crosstalk Provides Specific Exosomal Cargo to Direct Immune Response Licensing of Macrophages during Inflammatory Responses.

Neurodegenerative diseases (NDDs) continue to be a significant healthcare problem. The economic and social implications of NDDs increase with longevity. NDDs are linked to neuroinflammation and activated microglia and astrocytes play a central role. There is a growing interest for stem cell-based therapy to deliver genes, and for tissue regeneration. The promise of mesenchymal stem cells (MSC) is based on their availability as off-the-shelf source, and ease of expanding from discarded tissues. We tested the hypothesis that MSC have a major role of resetting activated microglial cells. We modeled microglial cell lines by using U937 cell-derived M1 and M2 macrophages. We studied macrophage types, alone, or in a non-contact culture with MSCs. MSCs induced significant release of exosomes from both types of macrophages, but significantly more of the M1 type. RNA sequencing showed enhanced gene expression within the exosomes with the major changes linked to the inflammatory response, including cytokines and the purinergic receptors. Computational analyses of the transcripts supported the expected effect of MSCs in suppressing the inflammatory response of M1 macrophages. The inflammatory cargo of M1 macrophage-derived exosomes revealed involvement of cytokines and purinergic receptors. At the same time, the exosomes from MSC-M2 macrophages were able to reset the classical M2 macrophages to more balanced inflammation. Interestingly, we excluded transfer of purinergic receptor transcripts from the co-cultured MSCs by analyzing these cells for the identified purinergic receptors. Since exosomes are intercellular communicators, these findings provide insights into how MSCs may modulate tissue regeneration and neuroinflammation.

Synergistic cytotoxicity of decitabine and YM155 in leukemia cells through upregulation of SLC35F2 and suppression of MCL1 and survivin expression.

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca2+/ROS-dependent manner, thereby promoting autophagy-mediated degradation of ß-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML.

Investigating influences of intravenous fluids on HUVEC and U937 monocyte cell lines using the magnetic levitation method.

Intravenous fluids are being widely used in patients of all ages for preventing or treating dehydration in the intensive care units, surgeries in the operation rooms, or administering chemotherapeutic drugs at hospitals. Dextrose, Ringer, and NaCl solutions are widely received as intravenous fluids by hospitalized patients. Despite their widespread administration for over 100 years, studies on their influences on different cell types have been very limited. Increasing evidence suggests that treatment outcomes might be altered by the choice of the administered intravenous fluids. In this study, we investigated the influences of intravenous fluids on human endothelial (HUVEC) and monocyte (U937) cell lines using the magnetic levitation technique. Our magnetic levitation platform provides label-free manipulation of single cells without altering their phenotypic or genetic properties. It allows for monitoring and quantifying behavior of single cells by measuring their levitation heights, deformation indices, and areas. Our results indicate that HUVEC and U937 cell lines respond differently to different intravenous fluids. Dextrose solution decreased the viability of both cell lines while increasing the heterogeneity of areas, deformation, and levitation heights of HUVEC cells. We strongly believe that improved outcomes can be achieved when the influences of intravenous fluids on different cell types are revealed using robust, label-free, and efficient methods.

Alternating electric fields can improve chemotherapy treatment efficacy in blood cancer cell U937 (non-adherent cells).

BACKGROUND: Recent achievements in cancer therapy are the use of alternating electrical fields at intermediate frequencies (100-300 kHz) and low intensities (1-3 V/cm), which specifically target cell proliferation while affecting different cellular activities depending on the frequency used. METHODS: In this article, we examine the effect of electric fields on spherical suspended cells and propose the combination of Daunorubicin, a chemotherapy agent widely used in the treatment of acute myeloid leukemia, with electric field exposure. U937 cells were subjected to an electric field with a frequency of 200 kHz and an intensity of 0.75 V/cm, or to a combination of Daunorubicin and electric field exposure, resulting in a significant reduction in cell proliferation. Furthermore, the application of an electric field to U937 cells increased Daunorubicin uptake. RESULTS: Apoptosis and DNA damage were induced by the electric field or in conjunction with Daunorubicin. Notably, normal cells exposed to an electric field did not show significant damage, indicating a selective effect on dividing cancer cells (U937). Moreover, the electric field affects the U937 cell line either alone or in combination with Daunorubicin. This effect may be due to increased membrane permeability. CONCLUSIONS: Our findings suggest that the use of electric fields at intermediate frequencies and low intensities, either alone or in combination with Daunorubicin, has potential as a selective anti-cancer therapy for dividing cancer cells, particularly in the treatment of acute myeloid leukemia. Further research is needed to fully understand the underlying mechanisms and to optimize the use of this therapy.

[Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells].

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

BCL2L1 inhibitor A-1331852 inhibits MCL1 transcription and triggers apoptosis in acute myeloid leukemia cells.

BH3 mimetics exert anticancer activity by inhibiting anti-apoptotic BCL2 proteins. However, accumulating evidence indicates that the off-target effects of these drugs tightly modulates their anticancer activities. In this study, we investigated whether the BCL2L1 inhibitor A-1331852 induced the death of U937 acute myeloid leukemia (AML) cells through a non-BCL2L1-targeted effect. A-1331852-induced apoptosis in U937 cells was characterized by increased ROS production, downregulation of MCL1, and loss of mitochondrial membrane potential. Ectopic expression of MCL1 alleviated A-1331852-induced mitochondrial depolarization and cytotoxicity in U937 cells. A-1331852-induced ROS production increased p38 MAPK phosphorylation and inhibited MCL1 transcription. Inhibition of p38 MAPK activation restored MCL1 expression in A-1331852-treated cells. A-1331852 triggered p38 MAPK-mediated Cullin 3 downregulation, which in turn increased PP2Acα expression, thereby reducing CREB phosphorylation. A-1331852 reduced the binding of CREB to the MCL1 promoter, leading to the inhibition of CREB-mediated MCL1 transcription. Furthermore, A-1331852 acted synergistically with the BCL2 inhibitor ABT-199 to induce U937 and ABT-199-resistant U937 cell death by inhibiting MCL1 expression. A similar phenomenon caused A-1331852-induced MCL1 downregulation and cytotoxicity in AML HL-60 cells. Collectively, our data suggest that A-1331852 shows an off-target effect of inhibiting MCL1 transcription, ultimately leading to U937 and HL-60 cell death.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

BACKGROUND: Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. METHODS: Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. RESULTS: Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. CONCLUSIONS: These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

Auraptene-induced cytotoxic effects in acute myeloid leukemia cell lines.

Acute myeloid leukemia is one of the most commonly identified hematological malignancies with poor prognosis. This research was planned to identify the cytotoxic effects of Auraptene on HL60 and U937 cell lines. The cytotoxic effects of Auraptene were measured by AlamarBlue assay (Resazurin) after 24- and 48-h treatments with different doses of Auraptene. The inductive effects of Auraptene on cellular oxidative stress were investigated by determining cellular ROS levels. The cell cycle progression and cell apoptosis were also evaluated by flow cytometry method. Our findings revealed that Auraptene decreased HL60 and U937 cellular proliferation by downregulation of Cyclin D1. Auraptene also induces cellular oxidative stress by upregulation of cellular ROS levels. Auraptene induces cell cycle arrest the early and late phases of apoptosis by upregulation of Bax and p53 proteins. Our data suggest that the anti-tumor function of Auraptene can be mediated by promoting apoptosis and cell cycle arrest and inducing cellular oxidative stress in HL60 and U937 cell lines. These results support that Auraptene may be used as a potent anti-tumor agent against hematologic malignancies in the further studies.

Nrf2-Mediated Pathway Activated by Prunus spinosa L. (Rosaceae) Fruit Extract: Bioinformatics Analyses and Experimental Validation.

In our previous studies, Prunus spinosa fruit (PSF) ethanol extract was showed to exert antioxidant, antimicrobial, anti-inflammatory and wound healing activities. In the present study, an integrated bioinformatics analysis combined with experimental validation was carried out to investigate the biological mechanism(s) that are responsible for the reported PSF beneficial effects as an antioxidant during a pro-inflammatory TLR4 insult. Bioinformatics analysis using miRNet 2.0 was carried out to address which biological process(es) the extract could be involved in. In addition, Chemprop was employed to identify the key targets of nuclear receptor (NR) signaling and stress response (SR) pathways potentially modulated. The miRNet analysis suggested that the PSF extract mostly activates the biological process of cellular senescence. The Chemprop analysis predicted three possible targets for nine phytochemicals found in the extract: (i) ARE signaling, (ii) mitochondrial membrane potential (MMP) and (iii) p53 SR pathways. The PSF extract antioxidant effect was also experimentally validated in vitro using the human monocyte U937 cell line. Our findings showed that Nrf2 is modulated by the extract with a consequent reduction of the oxidative stress level. This was confirmed by a strong decrease in the amount of reactive oxygen species (ROS) observed in the PSF-treated cells subjected to lipopolysaccharide (LPS) (6 h treatment, 1 µg/mL). No visible effects were observed on p53 and MMP modulation.

Legionella pneumophila Rhizoferrin Promotes Bacterial Biofilm Formation and Growth within Amoebae and Macrophages.

Previously, we showed that Legionella pneumophila secretes rhizoferrin, a polycarboxylate siderophore that promotes bacterial growth in iron-deplete media and the murine lung. Yet, past studies failed to identify a role for the rhizoferrin biosynthetic gene (lbtA) in L. pneumophila infection of host cells, suggesting the siderophore's importance was solely linked to extracellular survival. To test the possibility that rhizoferrin's relevance to intracellular infection was missed due to functional redundancy with the ferrous iron transport (FeoB) pathway, we characterized a new mutant lacking both lbtA and feoB. This mutant was highly impaired for growth on bacteriological media that were only modestly depleted of iron, confirming that rhizoferrin-mediated ferric iron uptake and FeoB-mediated ferrous iron uptake are critical for iron acquisition. The lbtA feoB mutant, but not its lbtA-containing complement, was also highly defective for biofilm formation on plastic surfaces, demonstrating a new role for the L. pneumophila siderophore in extracellular survival. Finally, the lbtA feoB mutant, but not its complement containing lbtA, proved to be greatly impaired for growth in Acanthamoeba castellanii, Vermamoeba vermiformis, and human U937 cell macrophages, revealing that rhizoferrin does promote intracellular infection by L. pneumophila. Moreover, the application of purified rhizoferrin triggered cytokine production from the U937 cells. Rhizoferrin-associated genes were fully conserved across the many sequenced strains of L. pneumophila examined but were variably present among strains from the other species of Legionella. Outside of Legionella, the closest match to the L. pneumophila rhizoferrin genes was in Aquicella siphonis, another facultative intracellular parasite of amoebae.

Polytetrafluorethylene microplastic particles mediated oxidative stress, inflammation, and intracellular signaling pathway alteration in human derived cell lines.

Microplastics (MPs) are now widely distributed across the aerial, terrestrial, and aquatic environments. Thus, exposure to MPs via the oral, inhalation, or dermal routes is inevitable. Polytetrafluoroethylene (PTFE)-MPs is mainly used for manufacturing nonstick cookware, semiconductors, and medical devices; however, their toxicity has been rarely studied. In the present study, six different human cell lines, which are representative of tissues and cells that directly or indirectly come into contact with MPs, were exposed to two different sizes of irregular shape PTFE-MPs (with an average diameter of 6.0 or 31.7 µm). PTFE-MPs-mediated cytotoxicity, oxidative stress, and changes in proinflammatory cytokine production were then evaluated. We found that the PTFE-MPs did not induce cytotoxicity under any of the experimental conditions. However, PTFE-MPs (especially average diameter of 6.0 µm) induced nitric oxide and reactive oxygen species production in all the cell lines tested. Moreover, both sizes of PTFE-MPs increased the secretion of tumor necrosis factor alpha and interleukin-6 from the U937 macrophage cell line and the A549 lung epithelial cell line, respectively. In addition, PTFE-MPs activated the MAPK signaling pathways, especially the ERK pathway, in A549 and U937 cells, and in the THP-1 dendritic cell line. We also found that the expression of the NLRP3 inflammasome was reduced in the U937 and THP-1 cell lines following treatment with the PTFE-MPs sized 31.7 µm average diameter. Furthermore, expression of the apoptosis regulator, BCL2, was markedly increased in the A549 and U937 cell lines. Thus, although PTFE-MPs exert different effects on different cell types, our findings suggest that PTFE-MPs-associated toxicity may be specifically linked to the activation of the ERK pathway, which ultimately induces oxidative stress and inflammation.

Inhibition of Cyclin-Dependent Kinases 8/19 Restricts Bacterial and Virus-Induced Inflammatory Responses in Monocytes.

Hyperactivation of the immune system remains a dramatic, life-threatening complication of viral and bacterial infections, particularly during pneumonia. Therapeutic approaches to counteract local and systemic outbreaks of cytokine storm and to prevent tissue damage remain limited. Cyclin-dependent kinases 8 and 19 (CDK8/19) potentiate transcriptional responses to the altered microenvironment, but CDK8/19 potential in immunoregulation is not fully understood. In the present study, we investigated how a selective CDK8/19 inhibitor, Senexin B, impacts the immunogenic profiles of monocytic cells stimulated using influenza virus H1N1 or bacterial lipopolysaccharides. Senexin B was able to prevent the induction of gene expression of proinflammatory cytokines in THP1 and U937 cell lines and in human peripheral blood-derived mononuclear cells. Moreover, Senexin B substantially reduced functional manifestations of inflammation, including clustering and chemokine-dependent migration of THP1 monocytes and human pulmonary fibroblasts (HPF).