Search Results (2,523)

Background: Prostate cancer (PCa) is the leading male urinary malignancy globally. Our previous article demonstrated the anti-PCa activity of Euphorbia humifusa Willd water extract (EHW) and some of its compounds via downregulating AR expression, but the anti-PCa active compounds from Euphorbia humifusa Willd (EH) and their mechanisms of action are yet to be clarified. Thus, the current article studied the in vitro anti-PCa effects of 3,3′-di-O-methylellagic acid (3,3′-di-O-Me-EA) derived from EHW and the related mechanism involved. Methods: 3,3’-di-O-Me-EA was isolated from EHW applying bioassay-guided fractionation. The spectroscopic methods were used to determining the structure of 3,3′-di-O-Me-EA. The drug-likeness and ADMET properties (absorption, distribution, metabolism, excretion, and toxicity) of 3,3′-di-O-Me-EA were analyzed in silico. Molecular docking and real-time surface plasmon resonance (SPR) analysis were performed to measure the interaction of 3,3′-di-O-Me-EA and VDAC1 protein. The viability and apoptosis of 22RV-1 and DU145 PCa cells were determined using MTT and Annexin V-FITC staining assay, respectively. q-PCR and Western blot experiments were used to analyzing the gene and protein expressions of VDAC1. Results: 3,3′-di-O-Me-EA was isolated and purified from EHW with a purity of ≥90.06%, and its structure was identified by HRTOF mass, NMR, and an authentic standard. In silico ADMET analysis indicated its favorable drug-like and pharmacokinetic properties. Molecular docking and SPR results confirmed that 3,3′-di-O-Me-EA could bind with the VDAC1 protein. Moreover, 3,3′-di-O-Me-EA dose- and time-dependently inhibited 22RV-1 and DU145 PCa cell viability, and induced apoptosis in a dose-dependent manner (p < 0.05). RT-qPCR and Western blot results showed that 3,3′-di-O-Me-EA dose-dependently up-regulated VDAC1 gene and protein expression levels in 22RV-1 and DU145 cells (p < 0.05). Meanwhile, in VDAC1-depleted 22RV-1 and DU145 cells, 3,3′-di-O-Me-EA down-regulated VDAC1 gene and protein expression levels, increased cell viability, and inhibited apoptosis compared to 22RV-1 and DU145 cells (p < 0.05). Furthermore, 3,3′-di-O-Me-EA enhanced VDAC1 gene and protein expression levels, inhibited cell viability, and induced apoptosis in VDAC1-overexpressed 22RV-1 and DU145 cells compared with 22RV-1 and DU145 cells (p < 0.05). Overall, EH active compound 3,3′-di-O-Me-EA may inhibit viability and induce apoptosis of 22RV-1 and DU145 PCa cells via up-regulating VDAC1 gene and protein expression levels. Conclusion: The results indicated that the 22RV1 and DU145 PCa cell viability inhibitory effects of 3,3′-di-O-Me-EA isolated from EH may be mediated by induction of apoptosis through up-regulation of VDAC1 gene and protein expression levels. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) is frequently characterized by notably elevated Ki-67 expression, a hallmark of uncontrolled rapid cell-cycle progression. However, the underlying mechanisms remain unclear, leading to limited therapeutic options. Methods: In this study, hub gene was identified through integrated bioinformatic analysis of public datasets (TCGA-BRCA and METABRIC). Subsequent functional validation was performed both in vitro and in vivo using siRNA-mediated knockdown and small-molecule inhibitors. Phenotypic effects—including cell viability, cell cycle distribution, DNA synthesis, and clonogenic survival—were comprehensively assessed using MTT assays, flow cytometry, EdU, and colony formation assays. Protein-level changes were confirmed by Western blotting and immunohistochemistry (IHC). To dissect the transcriptional regulation of the key hub gene TTK, we first predicted potential upstream transcription factors using the JASPAR database; binding specificity was then validated through in silico motif analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Results: The mitotic kinase TTK is significantly overexpressed in TNBC compared with non-TNBC breast cancers. Notably, TTK overexpression exhibited a strong positive correlation with elevated Ki-67 indices and reduced overall survival in TNBC patients. Functional validation demonstrated that pharmacological or genetic inhibition of TTK effectively induced G2/M cell-cycle arrest and potently suppressed TNBC proliferation in both in vitro cell cultures and in vivo xenograft models. Mechanistically, TTK overexpression stems from enhanced transcriptional initiation driven by the transcription factor NFYA binding to the CCAAT box in the TTK promoter—an interaction newly identified here. Concurrently, TTK blockade disrupted spindle assembly checkpoint (SAC) signaling via BUB1B/MAD1L1 downregulation, triggering mitotic arrest and catastrophe. Conclusions: Collectively, these findings establish TTK as a key cell-cycle regulator driving TNBC proliferation. More importantly, targeting mitotic control through TTK inhibition represents an efficient strategy to impede the aberrantly fast cell cycle progression in TNBC. Full article

Colorectal cancer (CRC), characterized by epidermal growth factor receptor (EGFR) overexpression, is often associated with poor prognosis and limited therapeutic response to conventional chemotherapy. In this study, we developed EGFR-targeted extracellular vesicles (EGFR-tEVs) by transiently engineering donor cells to display the GE11 peptide, aiming to enhance the precision of doxorubicin (Dox) delivery. The physicochemical properties of EGFR-tEVs were characterized using TEM, NTA, and Western blot. In vitro, EGFR-tEV-Dox exhibited increased cellular uptake in EGFR-overexpressing HCT-116 cells, leading to the activation of the p53-Bax-cleaved PARP1 apoptotic pathway. Notably, while Dox treatment induced p53 in normal colon fibroblasts (CCD18-Co), it did not trigger significant Bax activation or PARP1 cleavage, suggesting a preference for survival-related signaling in non-malignant cells. In a xenograft mouse model, EGFR-tEVs + Dox administration resulted in a 33.1% reduction in tumor volume and an 82.8% decrease in Ki-67 expression compared to the control group. These results indicate that transient receptor-mediated targeting enhances functional drug delivery to malignant tissues while minimizing pro-apoptotic induction in normal cells. Our findings suggest that EGFR-tEVs + Dox represents a balanced therapeutic strategy that improves antitumor efficacy with a favorable safety profile for EGFR-positive colorectal cancer. Full article

The global spread of COVID-19 led to the rapid authorization of remdesivir as the first antiviral therapy. However, accumulating clinical evidence has linked its use to cardiac adverse effects. Understanding the mechanisms underlying remdesivir-induced cardiotoxicity is critical for optimizing its clinical use and ensuring patient safety. This study investigates the electrophysiological and molecular features underlying remdesivir-induced cardiac toxicity in male and female guinea pigs, aiming to elucidate the sex-dependent differences in cardiac dysfunction and the role of mitochondria in mediating these effects. A cardiac injury model was established via intraperitoneal administration of remdesivir. In vivo telemetry and ex vivo electrocardiography were used for continuous monitoring of cardiac electrical activity, while optical mapping enabled the assessment of action potential parameters and conduction properties. The histopathological alterations and mitochondrial ultrastructure were examined by hematoxylin–eosin staining and transmission electron microscopy. ELISA and Western blot analyses were performed to explore the inflammatory signaling, apoptosis, and mitochondrial dynamics. Remdesivir induced distinct sex-specific patterns of cardiac toxicity. Compared with female guinea pigs, male guinea pigs had significantly more severe myocardial injury, which was characterized by extensive inflammatory cell infiltration, marked mitochondrial disruption, and a higher incidence of sustained ventricular tachyarrhythmia. Overall, remdesivir was associated with sex-dependent cardiac toxicity, accompanied by mitochondrial impairment and inflammatory activation. Male guinea pigs were more susceptible to electrophysiological instability and mitochondrial dysfunction. These findings highlight the importance of carefully evaluating remdesivir’s cardiac effects and support the need for individualized, sex-specific considerations in its clinical administration. Full article

Background: Midkine (MDK), a secreted heparin-binding growth factor, is involved in tumor progression and metastasis. While serum MDK is widely recognized as a potential prognostic biomarker for colorectal cancer (CRC), its specific functional role and underlying mechanisms in CRC development are not fully understood. Methods: The four publicly available CRC microarray datasets—GSE41258, GSE44076, GSE81558, and GSE117606—along with TCGA-COAD and TCGA-READ datasets and their associated clinical data were obtained. MDK expression was measured at both the mRNA and protein levels using quantitative real-time PCR (qRT-PCR) and Western blotting. To investigate its oncogenic functions, a comprehensive set of assays was performed: transwell and wound healing assays for invasion and migration; CCK-8 and colony formation assays for proliferation; and tail vein/spleen injection models combined with xenograft models to study metastasis and tumor growth in vivo. To uncover underlying mechanisms, Western blotting was used to examine the involvement of epithelial–mesenchymal transition (EMT) and the PI3K/AKT signaling pathway. Results: MDK is significantly overexpressed in CRC tissues and cells compared to normal tissues and cells. Notably, patients with high MDK levels show poorer overall survival (OS). Overexpression of MDK increases CRC invasion, migration, proliferation, and metastasis both in vivo and in vitro, while its knockdown reverses these effects. Mechanistically, MDK activates the PI3K/AKT pathway, leading to increased AP2A1 expression and promotion of EMT in CRC. Conclusions: MDK promotes invasion, migration, proliferation, metastasis, and EMT in CRC cells through the PI3K/AKT pathway by inducing AP2A1 expression, which could serve as a diagnostic marker. The PI3K inhibitor LY294002 significantly reduces AP2A1 levels and inhibits MDK-induced malignant behaviors. Targeting MDK-related signaling pathways may offer new strategies for CRC treatment. Full article

The hypoxic microenvironment plays a critical role in the progression of canine osteosarcoma (OSA) by promoting different cellular responses, including the release of extracellular vesicles (EVs). Given the clinical aggressiveness of canine OSA, the aim of this study was to evaluate the miRNAome profile in EVs released in vitro by four canine OSA cell lines under hypoxic conditions. In particular, for this study we used two commercial canine osteosarcoma cell lines (D17 and D22) and two primary osteosarcoma cell lines obtained in our laboratory (Penny and Wall). D17, D22, Penny, and Wall cell lines were cultured under normoxic and hypoxic conditions (200 µM CoCl₂) for 24 h. EVs were isolated by size-exclusion chromatography and characterized by nanoparticle tracking analysis and Western blotting. miRNAs extracted from EVs were then sequenced and analyzed using bioinformatics approaches. The most representative miRNAs were identified and validated by qPCR using the miRCURY LNA miRNA PCR assay. miRNome profiling identified 233 miRNAs differentially expressed in EVs across all analyzed cell lines. Among these, 94 miRNAs were detected exclusively under hypoxic conditions. From this subset, 43 miRNAs were selected for further validation by qPCR. The qPCR results showed that miR-222-3p and miR-186-5p were significantly downregulated in the Wall cell line under hypoxia (p ≤ 0.05). TargetScan and pathway enrichment analyses demonstrated that miR-186-5p regulates target genes involved in different cellular processes. In human osteosarcoma, low serum levels of miR-222-3p are associated with poor prognosis, while miR-186-5p is recognized as a key hypoxia-responsive miRNA. Collectively, these results suggest the potential of EV-associated miRNAs as biomarkers in canine OSA and support their relevance in translational and comparative oncology. Full article

(1) Background: Mild traumatic brain injury (mTBI), the most prevalent form of traumatic brain injury, often results from repetitive impacts to the head and is associated with long-term neurological impairment. The pathophysiology of mTBI is multifactorial and involves alterations in mitochondrial bioenergetics, a key determinant of neuronal function and survival. Although mitochondrial dysfunction is recognized as a hallmark of mTBI, its long-term effects on bioenergetics and the roles of regulatory cytosolic and mitochondrial proteins remain poorly understood. We hypothesized that repeated mTBI (rmTBI) induces sustained deficits in mitochondrial bioenergetics that are associated with long-term changes in key bioenergetic and other regulatory proteins. (2) Methods: Using the repeated CHIMERA injury model in adult male rats, randomly assigned to sham or rmTBI groups, we assessed mitochondrial respiration in isolated mitochondria and whole cerebral cortex homogenates using a Clark O₂ electrode and an Oroboros O₂k respirometer at time points ranging from 1 day to 2 months post-injury. Western blotting was performed for expression of regulatory proteins HKI, DRP1, MFN2, VDAC1, and ANT2. (3) Results: At 2 months post-rmTBI, respiration was faster and uncoupled, while ATP synthesis was significantly slowed compared with sham rats. This was accompanied by decreased expression of mitochondrial MFN2 and ANT2, by increased mitochondrial expression of DRP1, and by decreased translocation of HKI to mitochondria. There was no significant difference in VDAC1 expression. Earlier time points showed no significant differences in bioenergetics or protein expression, but neuro-inflammatory markers (GFAP and Iba1) were significantly elevated at these earlier time points of post-injury. (4) Conclusions: These findings indicate that rmTBI leads to a delayed long-term impairment of mitochondrial bioenergetics associated with alterations in proteins critical for bioenergetic regulation and mitochondrial control. This suggests a pathophysiologic mechanism for the persistent cognitive and behavioral deficits observed following rmTBI. Full article

A homologous classification for vespid venoms is missing. This study compared Polistes dominula and Vespula spp. venoms to evaluate their homology level. P. dominula and Vespula spp. extracts, including V. germanica, V. maculifrons, V. pensylvanica, V. alascensis, and V. squamosa in equal proportions, were generated from venom sacs and were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using Vespula-positive sera. Bands described as allergenic were excised and sequenced through Liquid Chromatography–Mass Spectrometry tandem analysis (LC-MS/MS) to confirm their identity. Phospholipase (group 1) and hyaluronidase (group 2) enzymatic activities were measured. Group 1 and 5 3-D structures and sequence identity were analyzed in silico. The results showed that the P. dominula and Vespula spp. venom extracts exhibit similar protein profiles and comparable allergen composition, with phospholipase and hyaluronidase activities. The structures of Pol d 1 and Ves v 1 and Pol d 5 and Ves v 5 were highly similar, and the identity levels were high across and within the Polistes and Vespula genera (≥50%). These results suggest the inclusion of venoms from Polistes and Vespula genera as candidates to create a new homologous group for wasp venoms and indicate that the currently described homologous groups require revision. Full article

Milk-derived exosomes (MDEs) are bioactive nanocarriers rich in microRNAs (miRNAs) that play critical roles in post-transcriptional regulation during neonatal development and immune adaptation. However, the dynamic changes in miRNA expression across lactation stages and their biological functions remain insufficiently explored. We hypothesized that the miRNA cargo of buffalo MDEs exhibits temporal specificity, thereby dynamically matching the immune requirements of the neonatal calves. Therefore, the present study aimed to systematically characterize the miRNA expression profiles of MDEs derived from colostrum, transitional milk, and mature milk. MDEs were isolated, purified using differential ultracentrifugation, and characterized via transmission electron microscopy, Western blotting, and nanoparticle-tracking analysis. A total of 370 miRNAs were identified in the MDEs, with 220 (59.5%) co-expressed across colostrum, transitional milk, and mature milk. Comparative analysis revealed that colostrum MDEs exhibited the greatest miRNA diversity. Expression patterns of miRNAs showed distinct stage-specific clustering as lactation progressed. Compared to mature milk, 100 differentially expressed miRNAs (DE-miRNAs) were identified in colostrum MDEs, including 39 upregulated and 61 downregulated miRNAs. Bioinformatics analyses indicated that predicted target genes were associated with transmembrane transport, immune response, cell development, and apoptosis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified pathways involved in immune regulation, inflammation, and apoptosis. Moreover, macrophages incubated with buffalo colostrum MDEs showed upregulation of proliferation-related genes and downregulation of pro-inflammatory factors, suggesting an anti-inflammatory effect through activation of the phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) signaling pathway. These findings offer new insights into miRNA profiles of buffalo MDEs across the early postpartum transition and provide a preliminary basis for exploring immunomodulatory potential of buffalo MDEs. Full article

Background: Lavandula stoechas has attracted increasing attention for its potential anticancer properties; however, evidence regarding its effects on apoptotic signaling across different tumor types remains limited. Methods: In this study, the effects of dry and fresh ethanol extracts of Lavandula stoechas L. subsp. stoechas (LsDE and LsFE) were investigated in MDA-MB-231 triple-negative breast cancer, RT4 bladder carcinoma, and T98G glioblastoma cell lines, providing a comparative evaluation of their apoptotic effects. Long-term proliferative capacity was assessed using clonogenic survival assays, while apoptosis-related responses were evaluated by Annexin V–FITC/propidium iodide staining, quantitative RT-PCR of BAX and BCL2 and Western blot analysis of Bax, Bcl-2, and cleaved PARP1. Results: Both extracts significantly reduced clonogenic survival in all tested cancer cell lines, with LsDE showing stronger inhibitory effects in RT4 and T98G cells. Annexin V/PI analysis revealed cell type-dependent response patterns. In MDA-MB-231 cells, both extracts increased the proportion of PI-positive cells, suggesting a loss of membrane integrity, whereas RT4 cells exhibited increased early apoptotic and membrane-compromised populations. In contrast, T98G cells showed comparatively limited changes associated with apoptosis. Transcriptional analysis demonstrated extract- and cell line-specific modulation of the BAX/BCL2 ratio. Western blot analysis further demonstrated activation of mitochondrial apoptotic signaling through coordinated regulation of Bax and Bcl-2 and increased PARP1 cleavage. LsFE showed the strongest apoptosis-associated changes in MDA-MB-231 cells, whereas LsDE showed stronger effects in T98G cells, while both extracts were effective in modulating these proteins in RT4 cells. Conclusions: These findings indicate that ethanol extracts of L. stoechas impair long-term proliferative capacity and induce tumor type-dependent modulation of apoptosis-related markers. This study provides an integrated experimental framework that combines clonogenic survival assays, apoptosis analyses, gene expression, and protein-level measurements, supporting further investigation of L. stoechas extracts in cancer research. Full article

Telomerase reverse transcriptase subunit (TERT) is a key factor involved in telomere maintenance and genome stability, and the decline in its expression is closely related to cellular senescence. In this study, we established TERT monoallelic knockout (TERT+/−) and TERT overexpression (TERT-Over) cell lines in porcine iliac artery endothelial cells (PIEC) using CRISPR/Cas9 and PiggyBac systems to compare the effects of TERT monoallelic knockout versus overexpression on cellular biology. TERT expression and telomere length were assessed via qPCR and Western blot analysis. Cellular proliferation and senescence were evaluated using CCK-8 assays, cell cycle analysis, and SA-β-gal staining. Furthermore, the expression of key genes involved in cell proliferation, metabolism, and related signaling pathways was quantified using q-PCR. The results showed that the TERT mRNA level and telomere length decreased in TERT+/− cells. Meanwhile, we also observed that TERT+/− cells exhibited G1 phase arrest in the cell cycle, with suppressed proliferation and increased SA-β-gal-positive cells. This was accompanied by downregulation of cell cycle and proliferation-related genes, including c-Myc, the E2F family, and Ki-67, as well as downregulation of cell metabolism-related genes, including HIF1α, HK2, GLUT1, the SMAD family, FOXO1, and ATF4. In addition, cytochrome C was downregulated, suggesting activation of mitochondrial apoptotic signaling. Together, these findings indicate impaired proliferative and metabolic activity and are consistent with cellular senescence associated with telomere shortening. In TERT-overexpressing cells, the TERT gene expression and telomere length increase, cell proliferation accelerates, and the survival rate significantly increases under H₂O₂ treatment. This indicated that the overexpression of TERT can enhance resistance to oxidative stress, thus showing a kind of anti-aging phenotype. In conclusion, TERT monoallelic knockout induces cellular senescence-associated phenotypes in porcine endothelial cells, whereas TERT overexpression enhances proliferation and resistance to oxidative stress under the experimental conditions used in this study. The two porcine cell models established here may provide useful experimental materials for studying aging-related mechanisms and evaluating anti-aging interventions in large animals. Further studies are needed to directly determine their effects on cellular replicative lifespan. Full article

In this study, the chemical composition of highland barley (HB), microwave fluidization HB (HB-1), extrusion and puffing HB (HB-2), and ultrafine pulverization HB (HB-3) were investigated based on untargeted metabolomics. In addition, RNA-seq transcriptomics, real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis were used to investigate the lipid metabolism mechanism of HB-1, induced by a high fat and cholesterol diet (HFCD). The results indicated that a total of 1292 metabolites were detected and classified into 78 distinct classes in the untargeted metabolomics analysis including fatty acyls, carboxylic acids and derivatives, glycerophospholipids, organooxygen compounds, prenol lipids, and so on. HB-1, HB-2, and HB-3 all increased the levels of amino acids and their derivatives, phenols, and carboxylic acid and its derivatives compared with HB. Furthermore, RNA-seq transcriptomic results indicated that HB-1 significantly modulated key genes of Cyp2c38, Cyp2b13, and Cyp2b9 related to steroid hormone biosynthesis and CD36, Plin4, and Fabp4 related to the PPAR signaling pathway, which played key roles in lipid metabolism. Moreover, qRT-PCR and WB results indicated that HB-1 obviously enhanced ADIPOQ expression level, while it reduced SCD-1, CD36, Fabp4, and SREBP-1c expression levels, suggesting that the alleviation of lipid metabolic dysregulation by HB-1 in hyperlipidemia mice might be mediated via participating in the PPARγ pathway. This study provided essential theoretical insights for the development and utilization of HB. Full article

Crohn’s disease (CD) is frequently accompanied by T-cell lymphopenia and impaired mucosal immunity, conditions that may predispose to intestinal microsporidiosis by Encephalitozoon cuniculi. This prospective case–control study examined the interplay between IL-7/IL-7 receptor (IL-7R) signaling and anti-E. cuniculi immune responses in 50 CD patients and 50 matched healthy controls. Serum IL-7 and anti-E. cuniculi IgG, IgM, IgA and IgE were quantified by ELISA, while intestinal expression of IL-7, CD127 (IL-7Rα) and CD132 (IL-7Rγ) was assessed by RT-PCR. Protein levels of IL-7 and caspase-3 were evaluated by Western blot, and lymphocyte subsets and apoptosis by flow cytometry. CD patients showed reduced anti-E. cuniculi IgG and IgM levels but increased seropositivity, indicating compromised humoral quality despite greater exposure. Compared with controls, CD was associated with decreased serum IL-7, increased mucosal IL-7, downregulated CD132, and diminished caspase-3, suggesting a disrupted IL-7/IL-7R-apoptosis pathway. In CD, IgA- and IgE-skewed responses correlated differentially with caspase-3 and CD56⁺ γδ T cells, while E. cuniculi seropositivity independently predicted a shorter surgery-free interval. These findings identify a profound dysregulation of the IL-7/IL-7R-CD132-caspase-3 axis in CD and implicate E. cuniculi exposure as a potential marker of impaired mucosal immunity and adverse outcomes. Full article

Background: Dodder, the dried mature seed of Cuscuta chinensis Lam. (CCL), has demonstrated anti-tumor activity, but its molecular and immunological mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear. Objective: To identify potential targets and elucidate the immune mechanisms by which CCL exerts therapeutic effects against ccRCC. Methods: A network pharmacology approach was employed to predict CCL’s bioactive components and their putative targets in ccRCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to explore relevant pathways. Molecular docking validated the binding of key compounds to hub proteins. In vitro assays—including cell viability, colony formation, invasion, and apoptosis measurements—assessed the effects of quercetin, a principal CCL constituent, on 786-O renal carcinoma cells. Flow cytometry was performed to determine the percentage of CD163⁺ cells. An in vivo xenograft model evaluated CCL’s anti-tumor efficacy. Western blotting, flow cytometry, and multiplex immunohistochemistry (mIHC) examined the modulation of signaling pathways and immune cell markers. Results: Network pharmacology identified IL-6, EGFR, TLR4, MMP9, CD44, and IFN-γ as core targets of CCL in ccRCC. Enrichment analyses implicated immune regulation, inflammation modulation, and PI3K/AKT signaling inhibition. Molecular docking revealed strong quercetin–MMP9 binding affinity. Immuno-correlation analyses indicated that high MMP9 levels positively correlated with macrophage infiltration and M2 polarization, suggesting a role in tumor immune escape. Quercetin significantly reduced the viability of 786-O cells in a dose-dependent manner, showing approximately 45% inhibition at 80 μM (p < 0.01). In addition, quercetin decreased MMP9 expression and reduced the proportion of CD163-positive macrophages. These effects were reversed by FSL-1 TFA (Toll-like receptor 2/6 agonist), which is the agonist of MMP-9. In the xenograft model, tumor volume in the quercetin-treated group was reduced by approximately 50% compared with the control group. Conclusions: CCL, particularly its active component quercetin, may inhibit ccRCC progression via inhibiting MMP9-mediated M2 macrophage polarization. Full article

Thyroid cancer (TC) is the most common endocrine malignancy, and challenges persist in preoperative diagnosis of indeterminate nodules and postoperative monitoring when thyroglobulin (Tg) assays are compromised by interfering anti-Tg antibodies (Tg-Ab). Extracellular vesicles (EVs) carry molecular cargo reflective of cells of origin and are increasingly explored as biomarker sources. In this study, we investigated whether thyroid-derived EVs retain the expression of thyroid-specific thyrotropin-receptor (TSHR), a suitable target in immunoaffinity-based EV isolation, and explored the presence of Tg in EV cargo as potential surrogate for serum Tg. EVs from thyroid cell lines (Nthy-Ori 3-1, TPC-1, OCUT2) and plasma of patients with benign, malignant tumors and recurrent TC were isolated by differential ultracentrifugation and characterized via nanoparticle tracking and Dot and Western blot analyses. EVs derived from Nthy-Ori 3-1 and TPC-1 cell lines were positive for surface TSHR and vesicular Tg, but not OCUT2. All plasma-derived EVs were positive for TSHR and Tg, while their electrophoretic profiles from vesicles differed compared to tissue lysate. Tg was detectable in EVs isolated from recurrent TC samples, even in Tg-Ab positive cases. Together, these results support the use of TSHR for targeted EV isolation and point to vesicular Tg as a potential recurrence marker. Full article