Biochemical and biophysical research communications最新文献

We previously showed that the benzoylphenylurea derivative BPU17 inhibits epithelial-mesenchymal transition and acts as an antifibrotic agent. This compound acts as a prohibitin (PHB) inhibitor by directly binding to PHB1. This binding disrupts the interaction between PHB1 and PHB2, leading to mild mitochondrial dysfunction. Here, we investigated the effect of BPU17 on angiogenesis using primary cultures of human vascular and microvascular endothelial cells, as well as a mouse model of choroidal neovascularization (CNV). A series of studies has shown that BPU17 inhibits angiogenesis both in vitro and in vivo. The molecular mechanism is that BPU17 inhibits serum response factor (SRF)/CArG box-mediated transcription by repressing the expression of SRF and its cofactor myocardin-related transcription factors (MRTF-A and -B [MRTF]). This defect causes the downregulation of adaptor and cell adhesion molecules such as vinculin and integrins, leading to the inhibition of angiogenesis. This inhibitory effect is closely associated with mild mitochondrial dysfunction, and siRNA-mediated knockdown of PHB1 similarly inhibits angiogenesis. Given that age-related inflammatory responses and subsequent choroidal neovascularization (CNV) contribute to the development of neovascular age-related macular degeneration (nAMD), this novel PHB inhibitor holds promise as a treatment for nAMD through its dual inhibitory effects on angiogenesis and fibrosis.

Senescence marker protein-30 (SMP-30; regucalcin) plays a crucial role in intracellular calcium homeostasis. This study investigated hepatic SMP-30 expression during non-alcoholic fatty liver disease (NAFLD) progression and evaluated the therapeutic potential of laminarin (LAM), a brown algae-derived polysaccharide, using high-fat diet (HFD)-fed mice and palmitic acid (PA)-treated Huh7 cells. Mice fed an HFD for 20 weeks developed NAFLD, characterized by elevated ALT/AST levels, hepatic steatosis, and significantly reduced SMP-30 expression. However, LAM treatment administered via drinking water (1%) or intraperitoneal injection (50 mg/kg) significantly attenuated lipid accumulation and restored hepatic SMP-30 expression. LAM reversed PA-induced lipid accumulation and SMP-30 downregulation in Huh7 cells. Mechanistically, LAM modulated the expression of SMP-30 and antioxidant proteins associated with activation of AKT/GSK3β/NRF2 signaling pathway, thereby mitigating the adverse effects of PA-induced toxicity. In conclusion, hepatic SMP-30 expression decreases during NAFLD progression, and LAM treatment restores these levels while alleviating lipid accumulation. These findings suggest that LAM may represent a promising therapeutic agent for NAFLD by improving lipid metabolism and reducing oxidative stress through the regulation of SMP-30.

This study aimed to investigate the ameliorative effect of hyperbaric oxygen (HBO) on a rat model of hyperuricemia induced by combined drugs and its underlying mechanism. A rat model of drug-induced hyperuricemia (HUA)was established by using a combination of yeast, potassium oxonate (PO) and hypoxanthine (Hx). During the modeling process, hyperbaric oxygen therapy was also administered. Serum uric acid (UA), creatinine (Cr)and blood urea nitrogen (BUN)levels were measured, and HE staining was used to evaluate the histopathological damage of small intestine and kidney tissues. Serum non-targeted metabolomics analysis was conducted, and 16 S rRNA gene sequencing was used to analyze the changes in intestinal flora structure, to evaluate the therapeutic effect of hyperbaric oxygen therapy. The results showed that HBO therapy could significantly reduce the UA, Cr and BUN levels and alleviate the histopathological damage of small intestine and kidney tissues in model rats. At the same time, HBO therapy could regulate the imbalance of intestinal flora, reduce potential pathogenic bacteria, and reduce the endogenous production of UA by decreasing the relative abundance of Bacteroides and Alistipes. Further serum metabolomics analysis indicated that HBO therapy could improve the differential metabolites in HUA rats, and the pathways of these differential metabolites were mainly related to glutathione metabolism, as well as d-glutamine and d-glutamate metabolism. HBO therapy could regulate key pathways such as purine metabolism and amino acid metabolism, and promote the generation of UA excretion-related metabolites. This study confirmed that HBO could improve combined drug-induced HUA by remodeling the intestinal flora structure and regulating the host metabolic pathways, providing new experimental evidence and potential targets for non-pharmacological treatment of HUA.

Hepatocellular carcinoma (HCC) remains a highly lethal malignancy despite therapeutic advances. SMARCB1 exhibits context-dependent functions across different cancer types. While it is frequently inactivated as a tumor suppressor in various malignancies, our previous work demonstrated that SMARCB1 acts as an oncogene in HCC by engaging the NUP210-P300 transcriptional axis. However, the upstream mechanisms that regulate SMARCB1 stability in HCC remain unexplored. Because SMARCB1 undergoes ubiquitin-mediated degradation under hypoxic conditions in other contexts, we investigated whether this regulation occurs in HCC. Cycloheximide (CHX) chase assays revealed that SMARCB1 remained highly stable under hypoxia in HCC cells, suggesting the presence of an active stabilization mechanism. Our screening for post-translational regulators identified USP21 as a deubiquitinase modulating SMARCB1 turnover. TCGA-LIHC analysis showed that USP21 is upregulated in HCC and positively correlated with SMARCB1 expression. Loss- and gain-of-function experiments confirmed that USP21 inhibition promotes SMARCB1 degradation, while USP21 overexpression prevents it. Co-immunoprecipitation demonstrated a physical interaction between USP21 and SMARCB1 that blocks ubiquitin-mediated proteasomal degradation. Combined inhibition of USP21 and P300, a downstream effector of SMARCB1, more effectively suppressed HCC cell proliferation under hypoxia than either treatment alone. Transcriptomic analysis further revealed that USP21-SMARCB1 axis contributes to immune-tolerant features in HCC. USP21 stabilizes SMARCB1 under hypoxic conditions, thereby sustaining its oncogenic and immunosuppressive activities in HCC. Targeting the USP21-SMARCB1 axis may inhibit tumor growth and enhance immunotherapy responsiveness, offering a potential therapeutic strategy for overcoming resistance in HCC treatment.

Resistance to lenvatinib has become a major obstacle in the clinical treatment of liver cancer, highlighting the significant research value and translational potential of developing synergistic drug combinations. In this study, deep learning models (MARSY and MatchMaker) were employed to predict potential synergistic partners for lenvatinib, with vincristine identified as a promising candidate. In vitro experiments confirmed that the combination synergistically inhibited the proliferation, migration, and clonogenic formation of liver cancer cells: CCK-8 and colony formation assays demonstrated a significant reduction in cell viability and clonogenic ability, while wound healing and Transwell assays indicated effective suppression of cell migration. The synergistic effect was quantitatively validated using the ZIP model. Furthermore, flow cytometry and Western blot analyses confirmed that the combination effectively induced apoptosis. Mechanistic studies revealed that the co-treatment led to excessive accumulation of intracellular reactive oxygen species (ROS), which activated the TNF-α/Caspase-8 signaling pathway, thereby inducing apoptosis in liver cancer cells. The cytotoxicity and pro-apoptotic effects were significantly attenuated by the ROS scavenger NAC. These findings provide a solid preclinical foundation for the further development of this combination therapy and underscore the importance of the "computational prediction-mechanistic validation" strategy in advancing cancer drug discovery.

We previously reported that murine fecal microRNAs (miRNAs) alter the composition of cultured gut microbiota and selectively increase Enterococcus. Since host miRNA expression is influenced by microbial colonization, this study investigated whether fecal miRNAs derived from germ-free (GF) and specific pathogen-free (SPF) mice differentially affect the composition and metabolic activity of cultured gut microbiota. Fecal miRNAs isolated from GF and SPF mice were added to cultures of murine gut microbiota. 16S rRNA gene sequencing showed that GF mice- and SPF mice-derived miRNAs altered microbial community structures in distinct ways. ANCOM identified Enterococcus as the only taxon significantly increased by miRNA treatment. GF mice-derived miRNAs significantly enhanced fermentation, as evidenced by a lower culture pH and higher concentrations of acetate, propionate, and lactate compared with SPF mice-derived miRNAs or vehicle controls. Correlation analysis demonstrated positive associations between organic acid levels and secondary fermenters, including Alloprevotella, Muribaculum, Lachnoclostridium, and Peptococcaceae. In contrast, typical saccharolytic butyrate producers such as Blautia, Lachnospiraceae, and Oscillospiraceae showed negative correlations. Microarray analysis revealed differences in the fecal miRNA profiles of GF and SPF mice, supporting the hypothesis that microbial exposure modulates fecal miRNA composition. Fecal miRNAs derived from antibiotic-treated mice did not reproduce the broad fermentation-promoting effects observed with GF mice-derived miRNAs, although lactate concentration increased. These observations suggest that fecal miRNAs promote fermentation by stimulating Enterococcus and downstream cross-feeding networks, and that prior microbial exposure attenuates this effect. Consequently, fecal miRNAs appear to represent a coevolved host mechanism that modulates microbial fermentation to maintain intestinal homeostasis.

Cardiac fibroblasts (CFs) are the predominant non-myocyte cell type in the heart and play central roles in extracellular matrix remodeling and intercellular signaling during cardiac physiology and pathology. However, the bioenergetic basis underlying CF functions remains poorly understood, mainly due to the lack of tools for visualizing intracellular adenosine triphosphate (ATP) dynamics with high spatiotemporal resolution. Here, we established immortalized murine cardiac fibroblasts stably expressing the genetically encoded ATP indicator GO-ATeam2 based on Förster Resonance Energy Transfer (FRET). The resulting CF7/GO-ATeam2 cell line allows real-time and quantitative monitoring of cytosolic ATP levels in living cells. CF7/GO-ATeam2 cells exhibited robust proliferation and quick responses to change of cytosolic ATP level. We demonstrated dynamic cytosolic ATP imaging upon pharmacological perturbations of oxidative phosphorylation and glycolysis, as well as under growth factor stimulation. Our work provides the CF7/GO-ATeam2 platform, a versatile cellular resource for dissecting the metabolic regulation of cardiac fibroblasts, offering new opportunities to explore energy dynamics in cardiac physiology and disease.

This study evaluates the radiobiological effectiveness comparing superficial X-rays to Cs-137 in V79 and U2OS cells cultured in T25 flasks using colony formation assays. Additional dosimetric care was given to maintaining equal absolute dose for all beam qualities, correcting for absorption and scattering in the flask, and quantifiable interpretation due to dose of the spatial differences in cell survival with significantly higher survival near the flask's edges. Ion chamber measurements quantified flask lid attenuation (6.9 %, 2.4 %, and 1.9 % for 50, 70, and 100 kVp), enabling dose correction for accurate survival analysis. CT-based Monte Carlo simulations revealed thicker flask walls and the meniscus effect created a 35 % dose reduction from flask center to edges, directly explaining the observed survival patterns. Monte Carlo simulation also predicted an increased secondary electron production at higher energies. These results emphasize the necessity of precise dosimetry in low-energy X-ray studies. Combined experimental and computational approaches enhanced reliability of radiobiological assessments. Survival curves yielded Relative Biological Effectiveness (RBE) values for 50 kVp X-ray of 1.45 (V79) and 1.64 (U2OS); 1.41 (V79) and 1.55 (U2OS) at 70 kVp; and 1.23 (V79) and 1.36 (U2OS) at 100 kVp, relative to Cs-137.

Chemotherapy is a significant treatment for cancer; however, it is frequently associated with deleterious side effects. Thymoquinone (TQ), a naturally occurring compound, has been reported to exhibit notable anti-cancer potential. The present study focuses on the in vitro effects of TQ-loaded nanoemulsion (TQ-NE) as an independent treatment and alongside the prevalent chemotherapeutic agents, 5-fluorouracil (5-FU) and capecitabine (CAP), on a colorectal cancer (HT-29) cell line. A TQ-NE formulation was synthesized and characterized, revealing an average particle size of 129.5 nm. The IC₅₀ values for CAP, 5-FU, TQ, NE (carrier), and TQ-NE were 39.57, 61.01, 21.81, 218.4, and 15.12 μM, respectively. Cytotoxic effects were examined in both HT-29 cells and fibroblasts, revealing that TQ-NE enhanced the sensitivity of cancer cells to chemotherapeutic agents. When combined with 5-FU or CAP, TQ-NE produced a greater reduction in cell viability than TQ alone, indicating synergistic interaction. Flow cytometry analysis further revealed that TQ-NE induced distinct apoptotic responses compared to the control, whereas its combination with chemotherapy drugs showed a different pattern. These findings suggest that TQ-NE can enhance the cytotoxic activities of 5-FU and CAP in vitro. Furthermore, using lower concentrations of TQ-NE could potentiate the inhibitory effects of 5-FU and CAP, presenting a promising strategy for improving colorectal cancer (CRC) treatment outcomes while potentially reducing the adverse effects related to higher doses of conventional chemotherapy.

Extracellular vesicles (EVs) are lipid bilayer-encased nano-size carriers that orchestrate molecular exchanges in the tumor microenvironment (TME) and carry significant information about the tumor development, progression and aggressiveness. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) mediates Helicobacter pylori adhesion to gastric epithelial cells and is markedly upregulated in gastric cancer (GC). This study identified that EVs secreted by H. pylori-infected gastric cancer cells (GCCs) were loaded with CEACAM6. In order to examine the tumorigenic potential of EVs released by H. pylori-infected cells with and without CEACAM6 overexpression, EVs were thoroughly characterised and several functional assays were conducted. CEACAM6 overexpressed cell-derived EVs mimicked the elevated status of CEACAM6 as in their source cells which were found to be further enhanced in EVs collected from infected cells. As revealed by the population-doubling, clonogenicity, wound-healing and matrigel invasion assays, CEACAM6-enriched EVs promoted oncogenic properties of recipient cells while EVs from H. pylori-infected CEACAM6-expressing cells further amplified these tumorigenic abilities. A novel approach of EV-sonication and fractionation identified that CEACAM6 were mainly located in the EV membrane. Interestingly, aligned with the finding of elevated CEACAM6 protein in the H. pylori infection-led metastatic GC tissue samples, sera from those GC patients exhibited significantly high CEACAM6 compared to those from the healthy volunteers. Collectively, these findings highlight CEACAM6-containing EVs as mediators of tumorigenesis. This study also showcases the technical, translational and clinical advantages of considering CEACAM6 as a diagnostic biomarker for the detection of GC in a minimally-invasive manner.