BACKGROUND:Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Gemcitabine (Gem) is a commonly used drug against HCC, but its efficacy is limited by the development of resistance. Resveratrol (Res), a natural polyphenol with antitumor activity, may reverse Gem resistance in HCC, although the mechanism remains unclear. METHODS:The effects of Res on the proliferation, apoptosis, cell cycle, and invasion of Hep3B and HuH-7 cells were assessed via cell counting kit-8 (CCK-8), clonogenic, flow cytometry, and Transwell assays, respectively. Potential Res targets were predicted by network pharmacology, and markers of HCC prognosis were identified from the cancer genome atlas (TCGA) data. The interaction between Res and thymidylate synthase (TYMS) was validated by molecular docking and dynamics simulation. A Gem-resistant HuH-7 cell line (HuH-7/GR) was established, and when these cells were treated with Res combined with Gem, the effect on Gem sensitivity was detected by CCK-8 assay, clonogenic assay, and flow cytometry. Finally, a subcutaneous nude mouse model of HCC was used to evaluate the in vivo effects of Res combined with Gem. RESULTS:Res inhibited HCC cell proliferation, induced apoptosis and G2/M arrest, and suppressed invasion in a concentration-dependent manner. Network pharmacology and TCGA analysis identified TYMS as an important target gene for Res. TYMS was highly expressed in HCC tissues and correlated with poor prognosis. Res treatment reduced TYMS expression, while molecular docking and simulation showed stable binding of Res to TYMS. TYMS levels were elevated in HuH-7/GR resistant cells. Res combined with Gem was found to reverse drug resistance, inhibit proliferation and colony formation, and induce apoptosis. The Res + Gem combination group showed the smallest tumor volume in the in vivo model. CONCLUSION:By attenuating Gem resistance through TYMS inhibition, Res holds promise as a clinically viable adjunct to Gem-based chemotherapy, offering a potential strategy to improve outcomes in HCC patients.
BACKGROUND:Multipotent mesenchymal stromal cells (MSCs) are widely used in regenerative medicine. Their decellularized extracellular matrix (dECM) has emerged as a key bioactive substrate for tissue engineering. The properties of MSC-derived dECM depend on cell culture conditions. Physiological hypoxia mimics the native MSC milieu and represents a promising preconditioning strategy. This study aimed to evaluate the structural and mechanical properties of dECM derived from MSCs cultured under different oxygen levels and to assess the phenotype of MSCs recellularized on these matrices. METHODS:Human adipose-derived MSCs were permanently expanded under 20% or 5% O2 to obtain 20-dECM and 5-dECM. Matrix architecture was visualized using scanning electron microscopy and quantitative phase contrast. Stiffness of dECM was measured by atomic force microscopy. MSCs were recellularized on dECM and cultured under 20% O2 for 72 hours. The assessment of cell morphology, cytoskeletal organization, nuclear translocation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), integrin expression, levels of reactive oxygen species (ROS), and cell cycle analysis were assessed using confocal microscopy and flow cytometry. RESULTS:In comparison with 20-dECM, 5-dECM demonstrated significant alignment of fibrillar bundles and a 1.6-fold increase in stiffness (p < 0.05). Reseeded MSCs on 5-dECM have acquired a spindle-shaped, aligned morphology, whereas cells on 20-dECM displayed a rounded morphology with multiple processes. The YAP and TAZ nuclear-to-cytoplasmic ratio was significantly lower on both dECM compared with uncoated surfaces (p < 0.0001). Total fluorescence intensity of YAP and TAZ per cell revealed divergent patterns on 5-dECM vs 20-dECM: YAP levels were increased, whereas TAZ levels were decreased. The expression of integrin α2 and α6 was higher (p < 0.01), while the expression of integrin α1 and intercellular adhesion molecule 1 (ICAM-1) was lower on both dECM vs control (p < 0.05). Intracellular ROS levels were reduced twofold on dECM (p < 0.01). The proportion of MSCs in the G2/M phase and the increase in cell number were more significant on dECM compared with uncoated plastic. CONCLUSIONS:Hypoxia-derived dECM exhibits increased anisotropy and stiffness, promoting MSC alignment and cytoskeleton organization, integrin expression, altering the subcellular localization of YAP and TAZ and reducing ROS levels. Thus, it may represent a promising, effective bioactive scaffold for MSC expansion.
BACKGROUND:Glioblastoma is the most aggressive primary malignant brain tumor of the central nervous system and remains difficult to treat because of rapid progression, diffuse invasion, and frequent recurrence. This study aimed to identify a potential natural compound against glioblastoma using a reverse network pharmacology strategy and to investigate its anti-glioma effects and underlying mechanism. METHODS:Glioblastoma-related targets were collected from public disease databases and integrated with differentially expressed genes from a public transcriptomic dataset to identify intersecting targets. Protein-protein interaction analysis, hub target screening, functional enrichment analysis, and reverse network pharmacology were performed to identify candidate compounds. Molecular docking and 100-ns molecular dynamics simulations were used to assess interactions between 20-hydroxyecdysone and pathway-related targets. Cell viability, colony formation, apoptosis, migration, and protein expression were examined in U251 and U87 glioma cells. Statistical differences were analyzed using a paired Student's t-test for two-group comparisons or one-way analysis of variance (ANOVA) followed by Dunnett's multiple-comparisons test for multi-group comparisons, as appropriate. RESULTS:A total of 136 intersecting targets and 18 hub targets were identified. Functional enrichment analysis indicated that the phosphatidylinositol 3-kinase/protein kinase B signaling pathway was one of the key enriched pathways. Reverse network pharmacology identified 20-hydroxyecdysone as a candidate compound. Molecular docking showed relatively strong binding of 20-hydroxyecdysone to epidermal growth factor receptor, fms-related receptor tyrosine kinase 1, and integrin subunit alpha 5, and molecular dynamics simulations supported the stability of these complexes. In vitro experiments showed that 20-hydroxyecdysone inhibited cell viability, clonogenicity, and migration, while promoting apoptosis. It also reduced the phosphorylation levels of phosphatidylinositol 3-kinase, protein kinase B, and glycogen synthase kinase 3 beta at Ser9 without markedly altering total protein expression. CONCLUSION:Treatment with 20-Hydroxyecdysone showed anti-glioma activity in vitro and may exert its effects, at least in part, through suppression of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway. These findings support its further evaluation as a potential therapeutic candidate for glioblastoma.
BACKGROUND:PDE4DIP encodes a scaffold protein that has been implicated in compartmentalized signaling and cytoskeletal organization, but the role of its myomegalin variant 8 (MMG8) isoform in hepatocellular carcinoma (HCC) remains unclear. To address this gap, we examined PDE4DIP expression in public HCC datasets and investigated the functional role of MMG8 in HCC models. METHODS:PDE4DIP expression was analyzed in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort and two Gene Expression Omnibus (GEO) cohorts (GSE14520, GSE36376). MMG8 function was assessed in Huh7 cells using siRNA-mediated knockdown and in Hepa1-6 cells using lentiviral Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated protein 9 (CRISPR-Cas9)-mediated knockout. Cell proliferation in MMG8-KD Huh7 cells and MMG8-KO Hepa1-6 cells was assessed using Cell Counting Kit-8 (CCK-8) assays, while Huh7 cell migration was evaluated using Transwell assays. Tumor growth was assessed using a murine subcutaneous tumor model. Immunohistochemical staining for Ki67 and cleaved caspase-3 was employed to assess tumor cell proliferation and apoptosis-associated changes, respectively. Gene set enrichment analysis was performed in TCGA-LIHC tumors stratified based on PDE4DIP expression. RESULTS:PDE4DIP expression differed between tumor and non-tumor tissues across HCC cohorts, although the directionality of this difference was not uniform. MMG8 knockdown in Huh7 cells reduced proliferation and migratory activity. A single-cell-derived MMG8-KO Hepa1-6 clone exhibited reduced proliferation in vitro and formed smaller tumors in vivo, with lower Ki67 positivity but no significant difference in cleaved caspase-3 positivity between groups. In tumors from the TCGA-LIHC cohort, PDE4DIP expression was associated with distinct transcriptional programs. Specifically, PDE4DIP-high tumors presented with positive normalized enrichment score (NES) values for several metabolic pathways, whereas adhesion/extracellular matrix (ECM), cell cycle/proliferation, and translation/ribosome-related pathways exhibited negative NES values. CONCLUSIONS:These findings support a functional contribution of MMG8 to proliferative, migratory, and tumor-growth phenotypes in the tested HCC models. Bulk gene-level PDE4DIP expression in human tumors was associated with context-dependent transcriptional states and should not be interpreted as a direct surrogate for MMG8 function.
