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Search Results (1,343)

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Keywords = cell-based functional assay

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17 pages, 11573 KiB  
Article
IFNγ Expression Correlates with Enhanced Cytotoxicity in CD8+ T Cells
by Varsha Pattu, Elmar Krause, Hsin-Fang Chang, Jens Rettig and Xuemei Li
Int. J. Mol. Sci. 2025, 26(14), 7024; https://doi.org/10.3390/ijms26147024 - 21 Jul 2025
Abstract
CD8+ T lymphocytes (CTLs) act as serial killers of infected or malignant cells by releasing large amounts of interferon-gamma (IFNγ) and granzymes. Although IFNγ is a pleiotropic cytokine with diverse immunomodulatory functions, its precise spatiotemporal regulation and role in CTL-mediated cytotoxicity remain incompletely [...] Read more.
CD8+ T lymphocytes (CTLs) act as serial killers of infected or malignant cells by releasing large amounts of interferon-gamma (IFNγ) and granzymes. Although IFNγ is a pleiotropic cytokine with diverse immunomodulatory functions, its precise spatiotemporal regulation and role in CTL-mediated cytotoxicity remain incompletely understood. Using wild-type and granzyme B-mTFP knock-in mice, we employed a combination of in vitro approaches, including T cell isolation and culture, plate-bound anti-CD3e stimulation, degranulation assays, flow cytometry, immunofluorescence, and structured illumination microscopy, to investigate IFNγ dynamics in CTLs. IFNγ expression in CTLs was rapid, transient, and strictly dependent on T cell receptor (TCR) activation. We identified two functionally distinct IFNγ-producing subsets: IFNγhigh (IFNγhi) and IFNγlow (IFNγlo) CTLs. IFNγhi CTLs exhibited an effector/effector memory phenotype, significantly elevated CD107a surface expression (a marker of lytic granule exocytosis), and higher colocalization with cis-Golgi and granzyme B compared to IFNγlo CTLs. Furthermore, CRTAM, an early activation marker, correlated with IFNγ expression in naive CTLs. Our findings establish a link between elevated IFNγ production and enhanced CTL cytotoxicity, implicating CRTAM as a potential regulator of early CTL activation and IFNγ induction. These insights provide a foundation for optimizing T cell-based immunotherapies against infections and cancers. Full article
(This article belongs to the Section Molecular Immunology)
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17 pages, 3907 KiB  
Article
Safety Validation of Plant-Derived Materials for Skin Application
by Euihyun Kim, Hyo Hyun Seo, Dong Sun Shin, Jihyeok Song, Seon Kyu Yun, Jeong Hun Lee and Sang Hyun Moh
Cosmetics 2025, 12(4), 153; https://doi.org/10.3390/cosmetics12040153 - 21 Jul 2025
Viewed by 48
Abstract
The cosmetic industry faces a critical need to balance commercial innovation with scientific validation, especially regarding the safety and efficacy of raw materials. Plant-derived materials (PDMs) offer a promising alternative to animal-derived ingredients in cosmetics, particularly due to their safety and compliance with [...] Read more.
The cosmetic industry faces a critical need to balance commercial innovation with scientific validation, especially regarding the safety and efficacy of raw materials. Plant-derived materials (PDMs) offer a promising alternative to animal-derived ingredients in cosmetics, particularly due to their safety and compliance with vegan and ethical standards. Unlike compounds such as polydeoxyribonucleotide (PDRN), which is derived from the testis or seminal fluid of Salmonidae species and raises concerns regarding its origin, sustainability, and consumer acceptability, PDMs provide a cleaner, ethically preferable profile. In this study, we evaluated 50 PDM candidates using in vitro cell viability, wound healing, and immunocytochemistry assays, along with primary skin irritation tests in human participants. None of the samples showed harmful effects. Notably, sample Nos. 38 and 42 demonstrated significant wound-healing capacity and upregulated filaggrin expression without causing notable irritation in clinical testing. These findings support the biological activity and safety of specific PDMs as functional cosmetic ingredients. This study presents scientifically validated evidence for plant-based alternatives to animal-derived materials and offers a new milestone in the shift toward sustainable and ethical cosmetic development. By bridging the gap between consumer demand and scientific rigor, this study provides a robust platform for future innovations in vegan cosmetics. Full article
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20 pages, 4705 KiB  
Article
GRK5 as a Novel Therapeutic Target for Immune Evasion in Testicular Cancer: Insights from Multi-Omics Analysis and Immunotherapeutic Validation
by Congcong Xu, Qifeng Zhong, Nengfeng Yu, Xuqiang Zhang, Kefan Yang, Hao Liu, Ming Cai and Yichun Zheng
Biomedicines 2025, 13(7), 1775; https://doi.org/10.3390/biomedicines13071775 - 21 Jul 2025
Viewed by 98
Abstract
Background: Personalized anti-tumor therapy that activates the immune response has demonstrated clinical benefits in various cancers. However, its efficacy against testicular cancer (TC) remains uncertain. This study aims to identify suitable patients for anti-tumor immunotherapy and to uncover potential therapeutic targets in TC [...] Read more.
Background: Personalized anti-tumor therapy that activates the immune response has demonstrated clinical benefits in various cancers. However, its efficacy against testicular cancer (TC) remains uncertain. This study aims to identify suitable patients for anti-tumor immunotherapy and to uncover potential therapeutic targets in TC for the development of tailored anti-tumor immunotherapy. Methods: Consensus clustering analysis was conducted to delineate immune subtypes, while weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, and support vector machine (SVM) algorithms were employed to evaluate the potential efficacy of anti-tumor immunotherapy. Candidate immunotherapy targets were systematically identified through multi-gene panel analyses and subsequently validated using molecular biology assays. A prioritized target emerging from cellular screening was further evaluated for its capacity to potentiate anti-tumor immunity. The therapeutic efficacy of this candidate was rigorously confirmed through a comprehensive suite of immunological experiments. Results: Following systematic screening of five candidate genes (WNT11, FAM181B, GRK5, FSCN1, and ECHS1), GRK5 emerged as a promising therapeutic target for immunotherapy based on its distinct functional and molecular associations with immune evasion mechanisms. Cellular functional assays revealed that GRK5 knockdown significantly attenuated the malignant phenotype of testicular cancer cells, as evidenced by reduced proliferative capacity and invasive potential. Complementary immunological validation established that specific targeting of GRK5 with the selective antagonist GRK5-IN-2 disrupts immune evasion pathways in testicular cancer, as quantified by T-cell-mediated cytotoxicity. Conclusions: These findings position GRK5 as a critical modulator of tumor-immune escape, warranting further preclinical exploration of GRK5-IN-2 as a candidate immunotherapeutic agent. Full article
(This article belongs to the Section Immunology and Immunotherapy)
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23 pages, 949 KiB  
Article
Anticancer Effect of Nature-Inspired Indolizine-Based Pentathiepines in 2D and 3D Cellular Model
by Roberto Tallarita, Federica Randisi, Lukas Manuel Jacobsen, Emanuela Marras, Mattia Riva, Giulia Modoni, Johannes Fimmen, Siva Sankar Murthy Bandaru, Carola Schulzke and Marzia Bruna Gariboldi
Cancers 2025, 17(14), 2393; https://doi.org/10.3390/cancers17142393 - 19 Jul 2025
Viewed by 242
Abstract
Background: 1,2,3,4,5-pentathiepines (PTEs) are compounds originally identified in marine ascidians and are currently under investigation for their promising pharmacological properties, particularly as potential antineoplastic agents. Objectives: In this study, we investigated the antineoplastic properties of a series of ten indolizine-based PTEs, comprising eight [...] Read more.
Background: 1,2,3,4,5-pentathiepines (PTEs) are compounds originally identified in marine ascidians and are currently under investigation for their promising pharmacological properties, particularly as potential antineoplastic agents. Objectives: In this study, we investigated the antineoplastic properties of a series of ten indolizine-based PTEs, comprising eight previously reported compounds and two newly synthesized derivatives. Methods: These compounds were evaluated against a panel of human cancer cell lines of diverse tissue origins, as well as, for the first time, on non-cancerous CR9 fibroblasts to assess their cytotoxic selectivity. In addition, their effects were tested on 3D spheroid models, providing preliminary insights into their potential in vivo efficacy. Initial screening focused on cell viability, followed by a more detailed characterization of the most active compounds in terms of their ability to induce apoptosis, necrosis, cell cycle arrest, and reactive oxygen species (ROS) generation. The anti-migratory activity of PTEs and a newly adapted assay to confirm sulfur species release in the cells were also performed for the first time. Results and Conclusions: Our findings reveal that four PTEs bearing hydrophilic, hydrogen-bonding functional groups, particularly the two inspired by natural analogs, exhibited the most potent anticancer activity. Full article
(This article belongs to the Special Issue Novel Therapeutic Approaches for Cancer Treatment)
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18 pages, 994 KiB  
Article
Optimizing PBMC Cryopreservation and Utilization for ImmunoSpot® Analysis of Antigen-Specific Memory B Cells
by Noémi Becza, Lingling Yao, Paul V. Lehmann and Greg A. Kirchenbaum
Vaccines 2025, 13(7), 765; https://doi.org/10.3390/vaccines13070765 - 19 Jul 2025
Viewed by 177
Abstract
Background: Measuring frequencies of antigen-specific memory B cells (Bmem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot® assays can provide [...] Read more.
Background: Measuring frequencies of antigen-specific memory B cells (Bmem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when Bmem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable Bmem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for Bmem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot® assays and increases the practical feasibility of extensive Bmem characterization in larger cohorts. Full article
(This article belongs to the Special Issue Vaccination-Induced Antibody and B Cell Immune Response)
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15 pages, 2992 KiB  
Article
Radiotherapy Upregulates the Expression of Membrane-Bound Negative Complement Regulator Proteins on Tumor Cells and Limits Complement-Mediated Tumor Cell Lysis
by Yingying Liang, Lixin Mai, Jonathan M. Schneeweiss, Ramon Lopez Perez, Michael Kirschfink and Peter E. Huber
Cancers 2025, 17(14), 2383; https://doi.org/10.3390/cancers17142383 - 18 Jul 2025
Viewed by 218
Abstract
Background/Objectives: Radiotherapy (RT) is a mainstay of clinical cancer therapy that causes broad immune responses. The complement system is a pivotal effector mechanism in the innate immune response, but the impact of RT is less well understood. This study investigates the interaction [...] Read more.
Background/Objectives: Radiotherapy (RT) is a mainstay of clinical cancer therapy that causes broad immune responses. The complement system is a pivotal effector mechanism in the innate immune response, but the impact of RT is less well understood. This study investigates the interaction between RT and the complement system as a possible approach to improve immune responses in cancer treatment. Methods: Human solid cancer (lung, prostate, liver, breast cancer), lymphoma, and leukemia cells were irradiated using X-rays and treated with polyclonal antibodies or anti-CD20 monoclonal antibodies, respectively. Chromium release assay was applied to measure cell lysis after radiation with or without complement-activating antibody treatment. The expression of membrane-bound complement regulatory proteins (mCRPs; CD46, CD55, CD59), which confer resistance against complement activation, CD20 expression, apoptosis, and radiation-induced DNA double-strand breaks (γH2AX), was measured by flow cytometry. The radiosensitivity of tumor cells was assessed by colony-forming assay. Results: We demonstrate that RT profoundly impacts complement function by upregulating the expression of membrane-bound complement regulatory proteins (mCRPs) on tumor cells in a dose- and time-dependent manner. Impaired complement-mediated tumor cell lysis could thus potentially contribute to radiotherapeutic resistance. We also observed RT-induced upregulation of CD20 expression on lymphoma and leukemic cells. Notably, complement activation prior to RT proved more effective in inducing RT-dependent early apoptosis compared to post-irradiation treatment. While complement modulation does not significantly alter RT-induced DNA-damage repair mechanisms or intrinsic radiosensitivity in cancer cells, our results suggest that combining RT with complement-based anti-cancer therapy may enhance complement-dependent cytotoxicity (CDC) and apoptosis in tumor cells. Conclusions: This study sheds light on the complex interplay between RT and the complement system, offering insights into potential novel combinatorial therapeutic strategies and a potential sequential structure for certain tumor types. Full article
(This article belongs to the Special Issue Combination Immunotherapy for Cancer Treatment)
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18 pages, 2563 KiB  
Article
The Potential Anti-Cancer Effects of Polish Ethanolic Extract of Propolis and Quercetin on Glioma Cells Under Hypoxic Conditions
by Małgorzata Kłósek, Anna Kurek-Górecka, Radosław Balwierz, Grażyna Pietsz and Zenon P. Czuba
Molecules 2025, 30(14), 3008; https://doi.org/10.3390/molecules30143008 - 17 Jul 2025
Viewed by 403
Abstract
Tissue hypoxia is commonly observed in head cancers and contributes to both molecular and functional changes in tumour cells. It is known to stimulate erythropoiesis, angiogenesis, and metabolic alterations within tumour cells. Glioblastoma, a type of brain tumour, is characterized by rapid proliferation [...] Read more.
Tissue hypoxia is commonly observed in head cancers and contributes to both molecular and functional changes in tumour cells. It is known to stimulate erythropoiesis, angiogenesis, and metabolic alterations within tumour cells. Glioblastoma, a type of brain tumour, is characterized by rapid proliferation and aggressive growth. Recent studies have indicated that natural products may hold potential as components of cancer therapy. Among these, Polish propolis and its active compound, quercetin, have demonstrated promising anti-cancer properties. The aim of this study was to evaluate the concentrations of selected cytokines—specifically IL-6, IL-9, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), interferon gamma-induced protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1)—produced by astrocytes of the CCF-STTG1 cell line. The cytotoxic effects of ethanolic extract of propolis (EEP) and quercetin were assessed using the MTT assay. Astrocytes were stimulated with lipopolysaccharide (LPS, 200 ng/mL) and/or IFN-α (100 U/mL), followed by treatment with EEP or quercetin (25–50 µg/mL) under hypoxic conditions for two hours. Cytokine concentrations were measured using the xMAP Luminex Multiplex Immunoassay and the Multiplex Bead-Based Cytokine Kit. Our study demonstrated that Polish propolis and its component quercetin modulate the tumour microenvironment in vitro, primarily by altering the levels of specific cytokines. The HCA analysis revealed that IL-6 and MCP-1 formed a distinct cluster at the highest linkage distance (approximately 100% of Dmax), suggesting that their expression patterns are significantly different from those of the other cytokines and that they are more similar to each other than to the rest. PCA analysis showed that EEP-PL (50 μg/mL) with IFN-α and EEP-PL (50 μg/mL) with LPS exert similar activities on cytokine secretion by astrocytes. Similar effects were demonstrated for EEP-PL 50 μg/mL + LPS + IFN-α, EEP-PL 25 μg/mL + IFN-α and EEP-PL 25 μg/mL + LPS + IFN-α. Our findings suggest that Polish propolis and quercetin may serve as promising natural agents to support the treatment of stage IV malignant astrocytoma. Nonetheless, further research is needed to confirm these results. Full article
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18 pages, 1756 KiB  
Article
ROR1 as an Immunotherapeutic Target for Inducing Antitumor Helper T Cell Responses Against Head and Neck Squamous Cell Carcinoma
by Ryosuke Sato, Hidekiyo Yamaki, Takahiro Inoue, Shota Sakaue, Hisataka Ominato, Risa Wakisaka, Hiroki Komatsuda, Michihisa Kono, Kenzo Ohara, Akemi Kosaka, Takayuki Ohkuri, Toshihiro Nagato, Takumi Kumai, Kan Kishibe, Hiroya Kobayashi and Miki Takahara
Cancers 2025, 17(14), 2326; https://doi.org/10.3390/cancers17142326 - 12 Jul 2025
Viewed by 287
Abstract
Background/Objectives: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer, with limited responsiveness to immune checkpoint inhibitors (ICIs). Cancer vaccine therapy is a promising novel immunotherapeutic approach that stimulates tumor-specific T cells. Receptor tyrosine kinase-like orphan receptor 1 [...] Read more.
Background/Objectives: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer, with limited responsiveness to immune checkpoint inhibitors (ICIs). Cancer vaccine therapy is a promising novel immunotherapeutic approach that stimulates tumor-specific T cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is overexpressed in malignant tumors but minimally expressed in normal tissues, presents a promising target for immunotherapy. This study aimed to evaluate ROR1 as a target for helper T lymphocyte (HTL)-based peptide vaccine immunotherapy in HNSCC. Methods: ROR1 expression in HNSCC tissues was assessed by immunohistochemistry. A novel ROR1-derived epitope (ROR1403–417) was identified and used to generate ROR1-reactive HTLs. Functional assays measuring IFN-γ and granzyme B secretion, as well as direct cytotoxicity, were performed. The effects of ICIs on HTL activity were also examined. The presence of ROR1-reactive T cells in the peripheral blood of patients with HNSCC was evaluated. Results: ROR1 positivity rates in HNSCC tissues were significantly higher (80.0%) than those in healthy controls (16.7%), and high ROR1 expression correlated with advanced clinical stages. HTL lines recognized the ROR1403–417 peptide in a human leukocyte antigen (HLA)-DR-restricted manner, secreted effector cytokines, and exhibited direct cytotoxicity against ROR1+ tumor cells. Dual PD-L1/PD-L2 blockade further enhanced HTL responses. ROR1-reactive T cells were detected in the peripheral blood of patients with HNSCC. Conclusions: ROR1 represents a promising target for immunotherapy in HNSCC. The ROR1403–417 peptide can elicit ROR1-reactive HTLs that exhibit antitumor responses against HNSCC cell lines, which can be enhanced by ICIs. These findings support the potential of ROR1-targeted peptide vaccine therapy for HNSCC. Full article
(This article belongs to the Section Clinical Research of Cancer)
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16 pages, 7688 KiB  
Article
Targeted Isolation of ω-3 Polyunsaturated Fatty Acids from the Marine Dinoflagellate Prorocentrum lima Using DeepSAT and LC-MS/MS and Their High Activity in Promoting Microglial Functions
by Chang-Rong Lai, Meng-Xing Jiang, Dan-Mei Tian, Wei Lu, Bin Wu, Jin-Shan Tang, Yi Zou, Song-Hui Lv and Xin-Sheng Yao
Mar. Drugs 2025, 23(7), 286; https://doi.org/10.3390/md23070286 - 10 Jul 2025
Viewed by 372
Abstract
In this study, we integrated HSQC-based DeepSAT with UPLC-MS/MS to guide the isolation of omega-3 polyunsaturated fatty acid derivatives (PUFAs) from marine resources. Through this approach, four new (14) and nine known (513) PUFA analogues [...] Read more.
In this study, we integrated HSQC-based DeepSAT with UPLC-MS/MS to guide the isolation of omega-3 polyunsaturated fatty acid derivatives (PUFAs) from marine resources. Through this approach, four new (14) and nine known (513) PUFA analogues were obtained from large-scale cultures of the marine dinoflagellate Prorocentrum lima, with lipidomic profiling identifying FA18:5 (5), FA18:4 (7), FA22:6 (8), and FA22:6 methyl ester (11) as major constituents of the algal oil extract. Structural elucidation was achieved through integrated spectroscopic analyses of IR, 1D and 2D NMR, and HR-ESI-MS data. Given the pivotal role of microglia in Alzheimer’s disease (AD) pathogenesis, we further evaluated the neuroprotective potential of these PUFAs by assessing their regulatory effects on critical microglial functions in human microglia clone 3 (HMC3) cells, including chemotactic migration and amyloid-β42 (Aβ42) phagocytic clearance. Pharmacological evaluation demonstrated that FA20:5 butanediol ester (1), FA18:5 (5), FA18:4 (7), FA22:6 (8), and (Z)-10-nonadecenoic acid (13) significantly enhanced HMC3 migration in a wound-healing assay. Notably, FA18:4 (7) also significantly promoted Aβ42 phagocytosis by HMC3 microglia while maintaining cellular viability and avoiding pro-inflammatory activation at 20 μM. Collectively, our study suggests that FA18:4 (7) modulates microglial function in vitro, indicating its potential to exert neuroprotective effects. Full article
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19 pages, 2082 KiB  
Article
De Novo Expressed Vpr Stimulates HIV-1 Replication in T Cells
by Blessing Enya and Jacek Skowronski
Viruses 2025, 17(7), 958; https://doi.org/10.3390/v17070958 - 7 Jul 2025
Viewed by 320
Abstract
Vpr, a virion-associated accessory virulence factor of HIV-1, promotes virus replication in both T cells and macrophages. Although Vpr’s early activity—antagonism of preintegration silencing and host restriction factors—has been documented, the relative contribution of virion-associated versus de novo expressed Vpr to HIV-1 replication [...] Read more.
Vpr, a virion-associated accessory virulence factor of HIV-1, promotes virus replication in both T cells and macrophages. Although Vpr’s early activity—antagonism of preintegration silencing and host restriction factors—has been documented, the relative contribution of virion-associated versus de novo expressed Vpr to HIV-1 replication fitness remains unclear. Here, we developed a T cell-based system that genetically separates early and late Vpr functions by combining tetracycline-inducible Vpr expression in CEM.SS T cells with vpr-deficient HIV-1 constructs and Gag p6 mutations that block Vpr packaging. CEM.SS T cells have been shown to recapitulate the positive effect of Vpr on HIV-1 replication observed in activated primary T cells. Using pairwise replication fitness assays under spreading infection conditions, we demonstrate that de novo synthesized Vpr exerts the dominant effect on HIV-1 replication in T cells, while virion-associated Vpr plays a lesser role. Somewhat unexpectedly, our findings reveal that antagonism of preintegration HIV-1 silencing by virion-associated Vpr is unlikely to be the major driver of enhanced HIV-1 replication in proliferating T cells. Instead, this function may play a more prominent role in the infection of non-dividing T cells and/or other cell types. Full article
(This article belongs to the Section Human Virology and Viral Diseases)
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14 pages, 1963 KiB  
Article
K562 Chronic Myeloid Leukemia Cells as a Dual β3-Expressing Functional Cell Line Model to Investigate the Effects of Combined αIIbβ3 and αvβ3 Antagonism
by Amal A. Elsharif, Laurence H. Patterson, Steven D. Shnyder and Helen M. Sheldrake
Methods Protoc. 2025, 8(4), 73; https://doi.org/10.3390/mps8040073 - 5 Jul 2025
Viewed by 656
Abstract
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 [...] Read more.
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
(This article belongs to the Special Issue Current Methodology Advances in Cell Therapy Applications)
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26 pages, 5282 KiB  
Article
Unraveling the Regulatory Impact of LncRNA Hnf1aos1 on Hepatic Homeostasis in Mice
by Beshoy Armanios, Jing Jin, Holly Kolmel, Ankit P. Laddha, Neha Mishra, Jose E. Manautou and Xiao-Bo Zhong
Non-Coding RNA 2025, 11(4), 52; https://doi.org/10.3390/ncrna11040052 - 4 Jul 2025
Viewed by 282
Abstract
Background/Objectives: Long non-coding RNAs (lncRNAs) play significant roles in tissue development and disease progression and have emerged as crucial regulators of gene expression. The hepatocyte nuclear factor alpha antisense RNA 1 (HNF1A-AS1) lncRNA is a particularly intriguing regulatory molecule in liver biology that [...] Read more.
Background/Objectives: Long non-coding RNAs (lncRNAs) play significant roles in tissue development and disease progression and have emerged as crucial regulators of gene expression. The hepatocyte nuclear factor alpha antisense RNA 1 (HNF1A-AS1) lncRNA is a particularly intriguing regulatory molecule in liver biology that is involved in the regulation of cytochrome P450 enzymes via epigenetic mechanisms. Despite the growing recognition of lncRNAs in liver disease, the comprehensive role of HNF1A-AS1 in liver function remains unclear. This study aimed to investigate the roles of the mouse homolog of the human HNF1A-AS1 lncRNA HNF1A opposite strand 1 (Hnf1aos1) in liver function, gene expression, and cellular processes using a mouse model to identify potential therapeutic targets for liver disorders. Methods: The knockdown of Hnf1aos1 was performed in in vitro mouse liver cell lines using siRNA and in vivo livers of AAV-shRNA complexes. Changes in the global expression landscapes of mRNA and proteins were revealed using RNA-seq and proteomics, respectively. Changes in the selected genes were further validated via real-time quantitative polymerase chain reaction (RT-qPCR). Phenotypic changes were assessed via histological and absorbance-based assays. Results: After the knockdown of Hnf1aos1, RNA-seq and proteomics analysis revealed the differential gene expression of the mRNAs and proteins involved in the processes of molecular transport, liver regeneration, and immune signaling pathways. The downregulation of ABCA1 and SREBF1 indicates their role in cholesterol transport and fatty acid and triglyceride synthesis. Additionally, significant reductions in hepatic triglyceride levels were observed in the Hnf1aos1-knockdown group, underscoring the impact on lipid regulation. Notably, the knockdown of Hnf1aos1 also led to an almost complete depletion of CYP7A1, the rate-limiting enzyme in bile acid synthesis, highlighting its role in cholesterol homeostasis and hepatotoxicity. Histological assessments confirmed these molecular findings, with increased hepatic inflammation, hepatocyte swelling, and disrupted liver architecture observed in the Hnf1aos1-knockdown mice. Conclusions: This study illustrated that Hnf1aos1 is a critical regulator of liver health, influencing both lipid metabolism and immune pathways. It maintains hepatic lipid homeostasis, modulates lipid-induced inflammatory responses, and contributes to viral immunity, indirectly affecting glucose and lipid metabolic balance. Full article
(This article belongs to the Section Long Non-Coding RNA)
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21 pages, 1507 KiB  
Article
Physicochemical Properties, Antioxidant and Antibacterial Activities and Anti-Hepatocarcinogenic Effect and Potential Mechanism of Schefflera oleifera Honey Against HepG2 Cells
by Jingjing Li, Jie Wang, Yicong Wang and Wenchao Yang
Foods 2025, 14(13), 2376; https://doi.org/10.3390/foods14132376 - 4 Jul 2025
Viewed by 405
Abstract
Schefflera oleifera honey (SH) is produced from the nectar of S. Oleifera by worker bees. Due to its unique properties and potential biological activities, this winter honey has attracted much attention. In this study, the physicochemical characteristics, antioxidant and antibacterial activities, antitumor effect [...] Read more.
Schefflera oleifera honey (SH) is produced from the nectar of S. Oleifera by worker bees. Due to its unique properties and potential biological activities, this winter honey has attracted much attention. In this study, the physicochemical characteristics, antioxidant and antibacterial activities, antitumor effect against HepG2 cells, and its potential mechanisms of SH were systematically evaluated. The results showed that different SH samples differed significantly in their physicochemical characteristics. The 910 chemical components, including 52 kinds of phenols, phenolic acids, and flavonoids, were detected in the methanol extract of SH using UHPLC-MS/MS by non-targeted metabolomics. Based on our limited knowledge, solanine and soyasaponin I are the first determined components in honey, and they may be used as characteristic substances of SH for identification and adulteration. SH had a weaker inhibitory effect against Salmonella typhimurium and Staphylococcus aureus than MH (UMF 10+), analyzed by MBC and MIC assays. Network pharmacology analysis showed that 95 overlapping targets were found between the active ingredients of SH and liver cancer cells (HepG2), which were enriched in KEGG of the PI3K-Akt pathway, Lipid and atherosclerosis, Proteoglycans in cancer, etc. The IC50 of SH against HepG2 cells was 5.07% (dw/v), which is lower than the glucose, fructose, and sucrose contents in SH on HepG2 cells, of 16.24%, 9.60% dw/v, and 9.94% dw/v, respectively. SH significantly down-regulated the expression of EGFR, AKT1, and SRC in HepG2 cells (p < 0.05), determined by an enzyme-linked immunosorbent assay kit, and induced cell cycle arrest and apoptosis by multiple pathways. These results provide a theoretical basis for its potential application in developing functional foods and additives. Full article
(This article belongs to the Section Food Physics and (Bio)Chemistry)
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30 pages, 4387 KiB  
Article
The Potential of Zanthoxylum acanthopodium DC. as Immunomodulators: Formulation, Activity Testing, and Extract Profiling
by Damaris Br. Hutapea, Yasmiwar Susilawati, Muhaimin Muhaimin, Riezki Amalia, Aisyah Tri Mulyani and Anis Yohana Chaerunisaa
Pharmaceuticals 2025, 18(7), 1001; https://doi.org/10.3390/ph18071001 - 3 Jul 2025
Viewed by 345
Abstract
Background/Objectives: One of the plants found in Indonesian forests that has potential as an herbal medicine is andaliman (Zanthoxylum acanthopodium DC.). The fruit of Z. acanthopodium contains phenolic compounds that are known to modulate the immune response. The purpose of this [...] Read more.
Background/Objectives: One of the plants found in Indonesian forests that has potential as an herbal medicine is andaliman (Zanthoxylum acanthopodium DC.). The fruit of Z. acanthopodium contains phenolic compounds that are known to modulate the immune response. The purpose of this study is to determine the extract profile and immunomodulatory activity of Z. acanthopodium fruit and to develop a soft capsule formulation of the extract in the form of emulsion, which stabilizes and acts as an immunomodulatory candidate. Methods: Extract profiling was conducted by liquid chromatography UHPLC–HRMS, and the predicted molecular structure was then used to search for the name of the compound using the mzcloud database. Immunomodulatory activity of the extract and its emulsion was assessed using a lymphocyte viability assay. The extract emulsion to be encapsulated as a soft capsule was developed by employing different types of oil and solubilizer in the oil phase, and a water phase containing the extract and two types of emulsifiers. Results: The chemical composition of andaliman extract was analyzed, including total phenolic content (4%), total flavonoid content (0.35%), and quercetin content (0.13%). Based on LC-HRMS analysis, eleven compounds derived from the ethanolic extract of andaliman were identified as potential immunomodulatory agents. The F3.3F formulation, which contains 30% MCT oil phase with solubilizer lauroyl-PEG-32 glycerides and a water phase with 35% Polysorbat (Tween) 80 emulsifier, provided the most stability. This stability is attributed to the presence of the Tween 80 emulsifier, which has superior wetting and washing functions, strong detergency, and good emulsifying properties compared to the PEG emulsifier used in formulation F3.3E. The survival rates in the lymphocyte cell viability test results indicate that treatment with andaliman extract (173.697% at 15.625 ppm; 174.923% at 31.25 ppm; 168.457% at 62.5 ppm) was better than treatment with kojic acid (144.375% at 15.625 ppm; 137.891% at 31.25 ppm; 146.345% at 62.5 ppm), used as the immunomodulatory agent standard. Conclusions: This study highlights the potential of andaliman extract as an immunomodulatory agent to be developed as an emulsion in a soft capsule. Full article
(This article belongs to the Section Natural Products)
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16 pages, 2188 KiB  
Article
Tartary Buckwheat Peptides Prevent Oxidative Damage in Differentiated SOL8 Cells via a Mitochondria-Mediated Apoptosis Pathway
by Yifan Xu, Yawen Wang, Min Yang, Pengxiang Yuan, Weikang Xu, Tong Jiang and Jian Huang
Nutrients 2025, 17(13), 2204; https://doi.org/10.3390/nu17132204 - 2 Jul 2025
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Abstract
Background: Under oxidative stress conditions, the increased levels of reactive oxygen species (ROS) within cells disrupt the intracellular homeostasis. Tartary buckwheat peptides exert their effects by scavenging oxidative free radicals, such as superoxide anion and hydrogen peroxide, thereby reducing oxidative damage within cells. [...] Read more.
Background: Under oxidative stress conditions, the increased levels of reactive oxygen species (ROS) within cells disrupt the intracellular homeostasis. Tartary buckwheat peptides exert their effects by scavenging oxidative free radicals, such as superoxide anion and hydrogen peroxide, thereby reducing oxidative damage within cells. Meanwhile, these peptides safeguard mitochondria by maintaining the mitochondrial membrane potential, decreasing the production of mitochondrial oxygen free radicals, and regulating mitochondrial biogenesis and autophagy to preserve mitochondrial homeostasis. Through these mechanisms, Tartary buckwheat peptides restore the intracellular redox balance, sustain cellular energy metabolism and biosynthesis, and ensure normal cellular physiological functions, which is of great significance for cell survival and adaptation under oxidative stress conditions. Objectives: In this experiment, a classical cellular oxidative stress model was established. Indicators related to antioxidant capacity and mitochondrial membrane potential changes, as well as pathways associated with oxidative stress, were selected for detection. The aim was to elucidate the effects of Tartary buckwheat oligopeptides on the metabolism of cells in response to oxidative stress. Methods: In this study, we established an oxidative damage model of mouse skeletal muscle myoblast (SOL8) cells using hydrogen peroxide (H2O2), investigated the pre-protective effects of Tartary buckwheat oligopeptides on H2O2-induced oxidative stress damage in SOL8 cells at the cellular level, and explored the possible mechanisms. The CCK-8 method is a colorimetric assay based on WST-8-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt], which is used to detect cell proliferation and cytotoxicity. Results: The value of CCK-8 showed that, when the cells were exposed to 0.01 mmol/L H2O2 for 1 h and 10 mg/mL Tartary buckwheat oligopeptides intervention for 48 h, these were the optimal conditions. Compared with the H2O2 group, the intervention group (KB/H2O2 group) showed that the production of ROS was significantly reduced (p < 0.001), the malondialdehyde (MDA) content was significantly decreased (p < 0.05), and the activity of catalase (CAT) was significantly increased (p < 0.01); the mitochondrial membrane potential in the KB/H2O2 group tended to return to the level of the control group, and they all showed dose-dependent effects. Compared with the H2O2 group, the mRNA expression of KEAP1 in the KB/H2O2 group decreased, while the mRNA expression of NRF2α, HO-1, nrf1, PGC-1, P62, and PINK increased. Conclusions: Therefore, Tartary buckwheat oligopeptides have a significant pre-protective effect on H2O2-induced SOL8 cells, possibly by enhancing the activity of superoxide dismutase, reducing ROS attack, balancing mitochondrial membrane potential, and maintaining intracellular homeostasis. Full article
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