Search Results (209)

Several of the integrin family of cell adhesion receptors have been popular targets for the development of anticancer agents, but with little clinical success to date. Cancer cells usually express multiple redundant integrins; one hypothesis for the lack of efficacy of current antagonists is their high selectivity for a single integrin. To address this, we developed a functional dual-β3-expressing cell model to investigate the effects of combined αIIbβ3/αvβ3 antagonism. We established that treating K562 chronic myeloid leukemia cells with 0.04 μM phorbol 12-myristate 13-acetate (PMA) for 40 h significantly upregulates functional αIIbβ3 and αvβ3 integrins. This optimized method provides a reliable platform for adhesion and detachment assays, enabling the characterization of dual integrin targeting strategies. Using this model, we demonstrate that combining αIIbβ3 and αvβ3 antagonists (GR144053 and cRGDfV) synergistically enhances inhibition of cell adhesion and promotes cell detachment compared to single-agent treatments. Our findings establish a reproducible approach for studying dual β3 integrin targeting, which can be used to investigate potential strategies for overcoming integrin redundancy in cancer therapeutics. Full article

Kujigamberol, a dinorlabdane compound isolated from Kuji amber, exerts multiple biological effects, including anti-allergic and anti-inflammatory activities. The present study demonstrated that kujigamberol inhibited cytokine production by T cells. In response to a phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM) stimulation, kujigamberol suppressed interferon-γ (IFN-γ) and interleukin-2 (IL-2) mRNA expression in murine T-cell lymphoma BW5147 cells stably transfected with the T-box transcription factor eomesodermin. IL-4 and Fas ligand mRNA expression was also inhibited by kujigamberol. In the murine cytotoxic T-cell line CTLL-2, kujigamberol more strongly decreased IFN-γ mRNA expression induced by IM alone than that induced by the combination of PMA and IM. A luciferase reporter assay showed that kujigamberol preferentially reduced nuclear factor of activated T cell (NFAT)-dependent transcription in human embryonic kidney 293T cells. Unlike the calcineurin inhibitor FK506, kujigamberol did not markedly affect NFATc2 protein levels in BW5147 cells but interfered with the binding of NFATc2 to the IFN-γ and IL-2 promoters. These results indicate that kujigamberol inhibited IFN-γ and IL-2 mRNA expression by preventing the binding of NFATc2 to their promoters; therefore, it has potential as an immunosuppressive agent. Full article

The Janus kinase 2 (JAK2) protein fulfills an important role in hematopoiesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, as it provides the genetic driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs), which are clinically manifested as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common cause of MPNs is the mutation of JAK2 V617F in the JAK2 gene, which results in increased cell proliferation. However, both the pathogenesis and treatment regimen of BCR::ABL1-negative MPNs remain poorly understood. The aim of the present study was to establish K562 cell lines with a point mutation in exon 14 (JAK2p.V617F) using CRISPR/Cas9 technology. The modified JAK2 V617F cell lines were examined for the gene mutation using droplet digital PCR (DDPCR), and the presence of the mutation was confirmed by DNA sequencing. Modified cells were characterized by measuring JAK2 gene expression and the extent of cell proliferation. Interferon α2a (IFN-α2a) and arsenic trioxide were also administered to the cells to explore their potential effects. The JAK2 V617F-mutated cells were found to exhibit a higher level of JAK2 gene expression compared with the wild type. Interestingly, a significant increase in the proliferation rate was observed with the modified cells compared with the wild type cells (p < 0.001), as assessed from the JAK2 gene expression levels. Furthermore, the treatments with IFN-α2a and arsenic trioxide led to the preferential suppression of the cell proliferation rate of the K562 expressing mutant JAK2 cells compared with the wild type cells, and this suppression occurred in a dose-dependent manner(p < 0.01). Moreover, the modified cells were able to differentiate into megakaryocyte-like cells following stimulation with phorbol 12 myristate 13 acetate (PMA). Taken together, the results of the present study have shown that the CRISPR/Cas9-modified JAK2 V617F model may be used as a disease model in the search of novel therapies for MPNs. Full article

Background: Chronic lymphocytic leukemia (CLL) is characterized by the proliferation of dysfunctional B cells, resulting in significant immune dysregulation. Patients with CLL exhibit varied responses to B cell receptor (BCR) targeted therapies, emphasizing the need for tailored immunotherapy approaches. This study investigated B cell function in untreated patients with CLL, and we further explored the effects of ex vivo protein kinase C activation on immune checkpoint expression and B cell profiles. Methods: Peripheral blood samples were collected from 21 untreated patients with CLL at King Edward Hospital in South Africa, between 2019 and 2022. B cells were stimulated with phorbol myristate acetate (PMA) and ionomycin. Using flow cytometry, the study explored the levels of B cell subsets and immune checkpoint proteins programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) expression on various B cell subsets. Results: PMA and ionomycin B cell stimulation upregulated PD-1, CTLA-4 and PD-L2 expression on B cell subsets (p < 0.01). As expected, monoclonal antibodies targeting PD-1, PD-L1 and CTLA-4 significantly downregulated the CTLA-4 expression of B cell subsets (p < 0.05), while PD-L2 exhibited varied responses in different B cell subsets. Moreover, PD-1 and PD-L1 expression on total B cells significantly declined following their blockage (p < 0.01). In addition, these monoclonal antibodies increased the levels of CD19⁺CD27⁺ B cells (p < 0.0128) and activated CD19⁺CD27⁺ B cells (p < 0.01). Conclusions: Protein kinase C activation on B cells stimulates immune checkpoint expression. The use of monoclonal antibodies on B cells plays a critical role in the B cell function through the reduction in CD38 expressing activated B cells and upregulation of CD19⁺CD27⁺ B cells. Moreover, the monoclonal antibody targeting PD-1, PD-L1 and CTLA-4 are effective in reducing the expression of CTLA-4 on B cell subsets, while PD-1 and PD-L1 blockage may be effective in reducing the expression of these immune checkpoints on total B cells. Full article

Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by fibrosis, immune dysregulation, and vascular abnormalities. Extracellular vesicles (EVs), secreted by immune cells, have been implicated in modulating fibroblast activity and are actively involved in SSc pathogenesis. This study aims to determine whether lymphomonocytic-derived EVs influence fibroblast proliferation and collagen synthesis in SSc. Fibroblasts from healthy donors (HDFs) and SSc patients (SScHDFs) were exposed to EVs derived from Jurkat and U937 cell lines stimulated under pro-inflammatory conditions using tumor necrosis factor-α (TNFα) or phorbol 12-myristate 13-acetate + ionomycin (PMA + IONO). Proliferation was assessed using CCK-8 assays, while collagen production was quantified via ELISA. Our findings demonstrate that EVs derived from PMA + IONO-stimulated Jurkat and U937 cells significantly reduced fibroblast proliferation in a dose-dependent manner. Notably, SScHDFs exhibited lower baseline proliferation and a diminished overall response to EV treatment. Collagen production was markedly reduced in both fibroblast types following exposure to PMA + IONO-stimulated EVs, whereas TNFα-stimulated EVs affected only HDFs. These findings suggest that EVs from activated immune cells modulate fibroblast function in SSc, potentially contributing to disease pathogenesis. Further research is warranted to elucidate the molecular mechanisms underlying these effects and to explore the therapeutic potential of targeting EV-mediated signaling in SSc. Full article

Background: Sparstolonin B (SsnB), a natural compound with anti-inflammatory and anti-proliferative properties, was investigated for its effects on cell viability, apoptosis, and inflammatory pathways in human colorectal cancer cells (HCT-116) and healthy human fibroblasts (BJ). Phorbol 12-myristate 13-acetate (PMA), a tumor promoter and inflammatory activator, was used to stimulate proliferation and inflammatory pathways. Methods: HCT-116 and BJ cells were treated with SsnB (3.125–50 μM) or PMA (1–10 nM) for 12–18 h. Cell viability was assessed using MTT analysis, while apoptosis was evaluated through cleaved caspase-3 staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry. Proliferation was analyzed through proliferating cell nuclear antigen (PCNA) staining. Toll-like receptor (TLR) signaling, cytokine expression, and sphingolipid levels were measured using immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, respectively. Results: SsnB reduced HCT-116 cell viability in a dose- and time-dependent manner with minimal effects on BJ cells. SsnB (25 μM, 12 h) decreased HCT-116 viability 0.6-fold, while PMA (10 nM, 12 h) increased it 2-fold (p < 0.01). No significant change was observed in BJ cells. PCNA fluorescence staining increased 2-fold with PMA and decreased 0.4-fold with SsnB (p < 0.001). PMA upregulated TLR2 and TLR4 mRNA and protein levels, with MyD88, p-ERK, and pNF-κB fluorescence increasing 2.1-, 1.5-, and 1.7-fold, respectively (p < 0.001). PMA elevated TNF-α, IL-1β, and IL-6 levels (p < 0.01). SsnB suppressed PMA-induced effects and promoted apoptosis, increasing cleaved caspase-3 levels by 1.5-fold and TUNEL staining by 1.9-fold (p < 0.01). Flow cytometry confirmed a significant increase in early and late apoptotic cells in the SsnB group. SsnB also increased ceramide (C18, C20, C22, and C24) levels (1.3- to 2.5-fold, p < 0.01) while reducing PMA-induced S1P and C1P increases (p < 0.01). Conclusions: SsnB selectively inhibits proliferation, induces apoptosis, and modulates inflammatory and sphingolipid pathways in colorectal cancer cells, with minimal toxicity to healthy fibroblasts, supporting its potential as a targeted therapeutic agent. Full article

This study aimed to investigate the innate immune response of human macrophages to Porphyromonas gingivalis W83 using a novel in vitro infection model. The growth kinetics of P. gingivalis W83 were analyzed, revealing an exponential growth phase at 8 h (optical density = 0.70). To establish a reliable macrophage model, the differentiation of THP-1 monocytes into macrophages was optimized using low concentrations of phorbol 12-myristate 13-acetate (PMA). This approach induced enhanced adherence and morphological changes, with full differentiation achieved after 48 h of PMA treatment followed by 24 h of rest. Polarization towards the pro-inflammatory M1 phenotype was successfully induced with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), as confirmed using cytokine profiling. Cytokine analysis using Luminex^® technology demonstrated significant increases in interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6, indicating the effective activation of macrophages towards a pro-inflammatory phenotype. Building upon this macrophage model, this study investigated the interactions between macrophages and P. gingivalis W83 during its exponential growth phase. After a one-hour infection period, bacterial DNA quantification in supernatants and lysed macrophages revealed minimal levels of internalized or adherent bacteria, supporting the hypothesis that P. gingivalis effectively evades immune detection. These findings emphasize the utility of this model in uncovering the sophisticated immune evasion strategies employed by P. gingivalis, with significant implications for the development of targeted therapeutic interventions. Full article

Dapsone is a sulfone used in treating inflammatory skin conditions. Despite its widespread dermatological use, the pharmacological actions of dapsone remain poorly understood. Here, we examined how different aspects of neutrophil functions are affected by dapsone. Peripheral blood neutrophils from healthy donors were stimulated with phorbol-12-myristate-13-acetate (PMA), N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), or calcium ionophore (CaI) or primed with cytokines prior to stimulation, in the presence of different concentrations of dapsone (from 10 to 50 µg/mL), followed by analyses of their survival, phenotype, and functional properties. We found that dapsone at the concentration of 50 μg/mL induced a significant neutrophil apoptotic rate during 6 h and 18 h, while other concentrations were well tolerated compared to control non-treated cells. However, dapsone significantly decreased the induced oxidative burst of neutrophils at all non-cytotoxic concentrations. Additionally, dapsone showed a dose-dependent suppression of NETosis in activated neutrophils. The production of IL-8 by dapsone-treated neutrophils was decreased under both stimulated (fMLP) and primed (TNF-α/fMLP) conditions. Moreover, dapsone inhibited the expression of CD11b/CD18, CD66, and CD89 and reversed or significantly mitigated the downregulation of CD16, CD32, CD181, CD88, and CD62L on neutrophils after priming and fMLP stimulation. In conclusion, our results indicate the complexity of dapsone actions on neutrophil functions, extending previous knowledge on the suppression of oxidative burst and IL-8 production upon neutrophils’ activation. Suppressed NETosis and modulation of marker expression associated with different neutrophil functions under inflammatory conditions are new findings, not recognized previously. Full article

Neutrophil extracellular traps (NETs) formation is a key process in inflammatory diseases like gout, but the underlying molecular mechanisms remain incompletely understood. This study aimed to establish a model to examine the formation of NETs induced by monosodium urate (MSU) and phorbol 12-myristate 13-acetate (PMA) and to elucidate their molecular pathways. Laser confocal microscopy was used to visualize NET formation, while flow cytometry was employed to detect reactive oxygen species (ROS) production. The microstructure of neutrophils was observed by transmission electron microscopy, and the expression of key proteins was determined by Western blotting. Additionally, the effect of various inhibitors targeting the MAPK signaling pathway on NET formation was evaluated. They include the Ras inhibitor Salirasib, Raf inhibitor Vemurafenib, ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, as well as NADPH oxidase inhibitor DPI and neutrophil elastase inhibitor Alvelestat. The results showed that MSU and PMA triggered significant NET formation, which was accompanied by increased ROS levels, lactate dehydrogenase release, dsDNA, and IL-8. Notably, selective MAPK pathway inhibitors and DPI and Alvelestat, except for SB203580, effectively down-regulated these indicators. These data indicated that the activation of a signaling pathway involving Ras-Raf-ERK, which is dependent on ROS, is crucial for the induction of NET formation by MSU and PMA. Given the involvement of NETs in multiple pathologies, our findings could potentially serve as molecular targets for the intervention and treatment of crystal-related diseases, especially for gout. Full article

The nociceptin receptor (NOP) and nociceptin are involved in the pathways of pain and inflammation. The potent role of nuclear factor-κB (NFκB) in the modulation of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β on the nociceptin system in human THP-1 cells under inflammatory conditions were investigated. Cells were stimulated without/with phorbol-myristate-acetate (PMA), TNF-α, IL-1β, or PMA combined with individual cytokines. To examine NFκB’s contribution to the regulation of the nociceptin system, PMA-stimulated cells were treated with NFκB inhibitor BAY 11-7082, JSH-23, or anacardic acid before culturing with TNF-α or IL-1β. NOP and prepronociceptin (ppNOC) mRNA were quantified by RT-qPCR; cell membrane NOP and intracellular nociceptin protein levels were measured by flow cytometry. Phosphorylation and localization of NFκB/p65 were determined using ImageStream. PMA + TNF-α decreased NOP mRNA compared to stimulation with PMA alone, while PMA + IL-1β did not. BAY 11-7082 and JSH-23 reversed the repression of NOP by PMA + TNF-α. TNF-α and IL-1β attenuated PMA’s upregulating effects on ppNOC. None of the inhibitors preserved the upregulation of ppNOC in PMA + TNF-α and PMA + IL-1β cultures. TNF-α strongly mediated the nuclear translocation of NFκB/p65 in PMA-treated cells, while IL-1β did not. Proinflammatory cytokines suppressed NOP and ppNOC mRNA in PMA-induced human THP-1 cells. NFκB signaling seems to be an important regulator controlling the transcription of NOP. These findings suggest that the nociceptin system may play an anti-inflammatory role during immune responses. Full article

Previous research has demonstrated that a combined magnetic field (CMF) plays a critical role in modifying the properties of aqueous solutions, leading to an increase in the luminol-enhanced chemiluminescence of neutrophils. Using this model, the distant interaction between aqueous solutions was demonstrated, and the role of a CMF in the regulation of this phenomenon was established. In the current study, highly diluted (HD) phorbol myristate acetate (PMA) solution (the donor) was incubated with aqueous ethanol (the acceptor), both in a CMF-generating device and under geomagnetic field (GMF), for 0, 20, and 60 min. After a 60 min incubation at a 0 cm distance with HD PMA under both GMF and CMF, acceptor samples added to neutrophils increased neutrophil chemiluminescence by approximately sevenfold. The ability of HD PMA, which had been incubated with an acceptor, to activate ROS production diminished within 60 min of observation. However, the HD PMA sample remained an effective donor for up to 6 days after preparation. At a 10 cm distance between the donor and acceptor, the activation of the acceptor did not occur. These findings provide new insights into the phenomenon of distant interaction of solutions, whose mechanisms are suggested to be related to the quantum electrodynamics of water molecular dynamic structures. Full article

Inflammation is a key process in the pathophysiology of various diseases, with macrophages playing a central role in the inflammatory response. This study investigates the anti-inflammatory potential of a newly synthesized analog of oleuropein (OP), the major olive tree (Olea europaea) metabolite. This derivative of OP, named Ole-Oxy, was designed by introducing an oxygen atom between the aromatic ring and the aliphatic chain of OP, to enhance interaction with proteins and improve bioactivity. Ole-Oxy demonstrated notable anti-inflammatory effects in vitro, particularly in phorbol 12-myristate 13-acetate-differentiated THP-1 macrophages, where it markedly reduced interleukin-6, tumor necrosis factor-α, and reactive oxygen species (ROS) levels, surpassing the effects of OP. In vivo, Ole-Oxy was evaluated in mouse models of acute skin and colon inflammation, showing significant efficacy in C57BL/6J mice, likely due to their Th1-biased immune response. Our results suggest that Ole-Oxy modulates inflammation through ROS scavenging and differential macrophage activation, underscoring the need for further research to fully elucidate its mechanism of action and optimize its pharmacokinetic properties for future therapeutic applications. Full article

Overactivation of the angiotensin-converting enzyme (ACE)/angiotensin II type 1 receptor (AT1R) pathway leads to vasoconstriction and elevated blood pressure. Persicaria minor (Huds.) Opiz is an herbal plant known for its antioxidant, anti-hyperlipidemic, and anti-atherosclerotic properties, with bioactive compounds that exhibit antihypertensive effects. Therefore, this study aimed to evaluate the antihypertensive effects of the standardized aqueous extract of P. minor leaf (AEPM) through the ACE/AT1R pathway in human umbilical vein endothelial cells (HUVECs) induced with phorbol 12-myristate 13-acetate (PMA). HUVECs were stimulated with PMA to induce ACE, with or without AEPM or captopril treatment, for 24 h. Subsequently, ACE mRNA expression, ACE protein levels, ACE activity, angiotensin II levels, and AT1R expression were measured. The results demonstrated that AEPM treatment significantly reduced ACE mRNA expression, ACE protein levels, ACE activity, angiotensin II levels, and AT1R expression in PMA-induced HUVECs. The modulatory effects of AEPM on the ACE/AT1R pathway were comparable to those of captopril. Ex vivo experiments further confirmed that AEPM reduced the contraction responses of rat aortic rings to PMA. In conclusion, P. minor effectively inhibits the ACE/AT1R pathway in PMA-induced HUVECs, suggesting its potential as a natural antihypertensive agent. Full article

Background: Neutrophils eject their DNA strings and cellular proteins into the extracellular space upon treatment with various stimulants. In the present study, we examined the effects of four major oxidized cholesterol metabolites on DNA release from granulocytes. Methods and Results: When oxysterols were added to HL-60-derived granulocytes stimulated with phorbol 12-myristate 13-acetate (PMA), they suppressed the release of DNA and myeloperoxidase from the cells. Among the four oxysterols tested, 7-ketocholesterol was the most effective. Addition of the same concentration of 7-ketocholesterol did not induce any cytotoxic effects, as evaluated based on the release of lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assays. DNA release from human peripheral blood neutrophils after PMA stimulation was also suppressed by 7-ketocholesterol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to quantify sterol content in the cells. The addition of oxysterols increased the cellular content of the corresponding compounds by more than 10-fold compared to those at baseline. Treatment of HL-60-derived granulocytes with methyl-β-cyclodextrin that removes sterol compounds from the membranes increased DNA release from the cells in a dose-dependent manner. Conclusions: These results suggest that oxysterols have suppressive effects on DNA release from granulocytes stimulated with PMA. Full article

Reduced/deficient expression of Protein Kinase C (PKC)ζ in Cord blood (CB) T cells is associated with allergy development in children and a propensity to maintain an immature T-helper (Th)2 cytokine profile. In addition, other PKC isozymes are also low in CBTCs. Since previous studies have reported that cord blood/neonatal monocyte and neutrophil functions are significantly lower than cells from adults, it was of interest to see if the CBTC PKC levels were reflected in CB monocytes and neutrophils. Compared to adult blood, CB expresses low levels of PKCα, β2, ε, θ, μ, ζ and λ/ι in monocytes and PKCα, β2, η, θ, μ, ζ and λ/ι in neutrophils. The T-cell PKCζ levels were positively correlated with levels in CB monocytes but not in neutrophils. However, neither the monocytes nor the neutrophil PKCζ were associated with T-cell development towards a Th1 or Th2 cytokine propensity, based on the production of interferon-gamma and interleukin-4 in response to phytohemagglutinin and phorbol myristate acetate. The results demonstrate that some newborn babies display a deficiency in PKC isozymes in monocytes and neutrophils, as reported for T cells. However, unlike T cells, the PKCζ levels of the phagocytes did not correlate with regulation of development towards a Th1 or Th2 cytokine phenotype. Full article