Biochemistry and Biophysics Reports

In TMPRSS2 fusion-positive prostate cancer, ERR is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERR binds to the ERREs (estrogen-related receptor response elements) present at -5042 bp of the TMPRSS2- promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERR recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERR (ERRFL), NTD deleted construct (ERR{Delta}NTD), and the DNA-binding domain (ERRDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5' -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3'). Circular dichroism analysis showed that all three ERR proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRa proteins. Binding analysis showed that ERRDBD bound weakly to the DNA, whereas ERRFL and ERR{Delta}NTD exhibited substantially higher affinities ~120-fold and ~131-fold than ERRaDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRFL structure and an ERRFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRa proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRa proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERR recognition of the TMPRSS2- promoter DNA, which provides insight into ERR-mediated transcriptional regulation and development of anticancer drugs against ERR-driven prostate cancer.

Background Zinc finger protein 36-like 1 (ZFP36L1) is an AU-rich element-binding RNA-binding protein that regulates post-transcriptional gene expression and has been implicated in tumor progression, cell-cycle regulation, and DNA damage responses. However, its functional role in triple-negative breast cancer (TNBC) remains poorly understood. This study investigated the effects of CRISPR/Cas9-mediated ZFP36L1 knockout on cell proliferation, doxorubicin (DOX) sensitivity, cell-cycle progression, and DNA damage responses in MDA-MB-231 TNBC cells. Methods Wild-type (WT) and CRISPR/Cas9-generated ZFP36L1 knockout (KO) MDA-MB-231 cells were cultured under standard conditions. Cellular proliferation was evaluated by cell counting over three weeks. Cell viability following DOX treatment was determined using the MTT assay, and half-maximal inhibitory concentration (IC50) values were calculated. Cell-cycle distribution was assessed by propidium iodide flow cytometry after 24 h of DOX exposure, while DNA damage was quantified by {gamma}-H2AX flow cytometric analysis. Statistical significance was determined using Student's t-test with P < 0.05 considered significant. Results ZFP36L1 knockout reduced the proliferative capacity of MDA-MB-231 cells compared with WT cells. Both cell lines exhibited dose-dependent decreases in viability following DOX treatment. KO cells demonstrated a higher mean IC50 than WT cells (9.64 vs. 8.40 M), indicating a trend toward reduced DOX sensitivity; however, this difference was not statistically significant (P = 0.569). Flow cytometric analysis revealed enhanced accumulation of KO cells in the S and G2/M phases following DOX treatment, suggesting altered cell-cycle checkpoint regulation. Furthermore, KO cells exhibited elevated basal {gamma}-H2AX expression and greater DOX-induced {gamma}-H2AX accumulation than WT cells, indicating increased DNA damage and impaired maintenance of genomic stability. Conclusions CRISPR/Cas9-mediated loss of ZFP36L1 suppresses proliferation, alters cell-cycle checkpoint dynamics, and enhances DNA damage accumulation in MDA-MB-231 TNBC cells. These findings indicate that ZFP36L1 plays a context-dependent role in regulating genomic stability and cellular responses to genotoxic stress, highlighting its potential as a biomarker and therapeutic target in triple-negative breast cancer.

Lung cancer continues to be the leading cause of cancer-related mortality globally, with non-small cell lung cancer (NSCLC) representing the most prevalent subtype. Tumor hypoxia is a characteristic feature of the neoplastic microenvironment in NSCLC, facilitating tumor progression and conferring resistance to oxidative stress through the stabilization of hypoxia-inducible factor-1 alpha (HIF-1). In this study, we investigated the combined anticancer effects of baicalein (Bai), a natural flavonoid, and thermal-cycling stimulation (TCS), a physical treatment that minimizes damage to normal cells, under cobalt (II) chloride (CoCl2)-induced hypoxic conditions in NSCLC. In A549 NSCLC cells, the combination of Bai and TCS significantly decreased cell viability and induced apoptosis, while exhibiting minimal cytotoxicity on IMR-90 normal human lung fibroblast cells. On a mechanistic level, this combined treatment suppressed the expression of HIF-1 and superoxide dismutase 2 (SOD2) proteins, elevated intracellular reactive oxygen species (ROS) levels, and impaired DNA repair capability by downregulating MutT homolog 1 (MTH1) protein expression. Additionally, disruption of mitochondrial membrane potential and increased poly (ADP-ribose) polymerase (PARP) cleavage further confirmed the induction of apoptosis. These findings indicate that combining Bai with TCS offers a promising synergistic approach to treating NSCLC under hypoxic conditions.

Melittin, a highly active natural antimicrobial peptide derived from honeybee venom, holds immense pharmacological potential against solid tumors. However, its specific anti-tumor efficacy and transcriptomic dynamics in cervical carcinoma remain to be systematically characterized. This study evaluated the anti-cancer properties of melittin on murine U14 cervical cancer cells following by transcriptomic investigation of the underlying mechanism. Phenotypic evaluations revealed that melittin potently inhibited U14 cell viability, while wound healing assays demonstrated a profound, dose-dependent suppression of cellular migration, culminating in near-complete migratory arrest at high concentrations. Furthermore, flow cytometry quantified a dramatic, dose-dependent surge predominantly in late apoptotic cell populations. These apoptotic events were structurally corroborated by scanning electron microscopy (SEM), which revealed severe plasma membrane perforation and morphological exhaustion. Enrichment analyses indicated that the physical membrane disruption inflicted by melittin translated into a severe metabolic crisis, marked by a global suppression of ribosomal biogenesis and mitochondrial oxidative phosphorylation. Furthermore, melittin profoundly repressed the Tie2-mediated angiogenic pathway (Etv2 down-regulation) while triggering the lethal hyperactivation of the AP-1 transcriptional stress complex (Jun, Fos, Fosb). Collectively, these findings elucidate the pharmacological networks underlying melittins cytotoxicity, providing solid molecular evidence for its development as a natural therapeutic agent against cervical cancer.

Objective: Preclinical studies suggest that irradiation combined with anti-PD-1 antibody (iRT) exacerbates cardiac injury in mice, partly through CD8 T-cell-mediated mechanisms. We investigated radiation-induced PD-L1 expression in cardiomyocytes and explored its potential role in iRT-associated cardiac injury. Methods: AC16 cells were exposed to different doses of irradiation. PD-L1 expression was assessed at different time points by flow cytometry, qRT-PCR, western blotting, and immunofluorescence. Cell viability, apoptosis, and cytokine levels were evaluated. PD-L1 was knocked down using siRNA to assess its function. PBMCs or CD8 T cells were co-cultured with irradiated or non-irradiated AC16 cells at different E:T ratios, with or without PD-1 blockade. A C57BL/6J mouse model of iRT-induced myocardial injury was established, and myocardial PD-L1 expression and T-cell infiltration were evaluated. Results: Irradiation upregulated PD-L1 expression in AC16 cells, with increased membrane and cytoplasmic signals at 48 h and prominent nuclear-associated fluorescence at 72 h. Irradiation increased IL-6, MCP-1, CCL5, and CXCL10 release, decreased IL-10, reduced cell viability, and increased apoptosis. PD-L1 knockdown further increased inflammatory cytokine and chemokine release but did not significantly alter apoptosis or further reduce viability in irradiated AC16 cells. In the presence of a high density of activated CD8 T cells, disruption of PD-1/PD-L1 signaling was associated with a further reduction in AC16 cell viability. In vivo, myocardial PD-L1 expression was increased, whereas CD8 T-cell infiltration remained limited and focal. Conclusion: PD-L1 upregulation in cardiomyocytes may exert an adaptive protective and immunoregulatory role during iRT-associated injury, potentially by limiting inflammatory mediator release and modulating susceptibility to activated CD8 T-cell mediated injury. CD8 T-cell density and activation are the key issues.

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp2 group, providing promising candidates for further therapeutic development.

Boron neutron capture therapy (BNCT) is one of the promising treatment methods for cancer. BNCT is based on the unique high ability of the non-radioactive boron-10 (10B) nucleus to absorb thermal neutrons. The absorption of a neutron by boron results in a nuclear reaction 10B(n,)7Li with 84% of the energy being released within the cell, thereby inducing cancer cell death. We propose a novel approach based on using lithium instead of boron in neutron capture therapy: the neutron capture reaction cross-section for lithium is 4 times smaller than that of boron, while the energy release is 2 times higher, and the primary advantage of lithium is that 100% of the energy is released inside the cell. A series of in vivo neutron irradiation experiments using the murine B16 skin melanoma model demonstrated the efficacy of lithium neutron capture therapy (LiNCT). Higher lithium concentrations in the tumor achieved through intraperitoneal administration, compared to the oral route, contributed to a significant increase in animal survival and a significant decrease in tumor growth. The results of our study provide entirely new opportunities for the development of neutron capture therapy, both with lithium and with a possible lithium-boron combination.

Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8) is believed to function as a tumor driver; however, to understand its molecular characteristics across tumors, a comprehensive pan-cancer analysis of EPS8 is lacking. Objective: This study aimed to investigate the prognostic, molecular, and immunological significance of EPS8 across multiple human cancers. Methods: Comprehensive analyses were conducted using GEPIA2, UALCAN, TIMER2.0, cBioPortal, SMART, GSCA, Enrichr, and the TCGAplot R package to evaluate survival prognosis, gene expression, DNA methylation, immune infiltration, proteomic expression, tumor mutational burden (TMB), microsatellite instability (MSI), genetic alterations, drug sensitivity, and enriched pathways. Results: EPS8 overexpression in LGG (p=7.2x10-5) and PAAD (p=9.4x10-6) was significantly associated with poor overall survival and disease-free survival. Amplification was the most common genetic alteration, with alteration frequencies approaching 6% in TGCT and UCEC. In KIRC and PAAD, EPS8 expression correlated with tumor stage. Immune infiltration analysis revealed significant associations between EPS8 expression and immune cells. EPS8 expression also showed significant correlations with both TMB and MSI in STAD and ESCA. Enrichment analysis indicated an association between EPS8 and regulation of the actin cytoskeleton and similar pathways. Conclusion: These findings suggest that EPS8 has prognostic and immunological significance across multiple cancer types and may serve as a potential biomarker. The observed associations with immune-related features and drug response provide a basis for future experimental studies to evaluate its role in cancer biology and its potential relevance to immunotherapy.

RationaleOxidative stress, resulting from an imbalance between the production of reactive oxygen species (ROS) and antioxidant defence mechanisms, disrupts cellular redox homeostasis and contributes to damage of biomolecules, including nucleic acids, proteins and lipids. Such disturbances can impair intracellular signalling pathways and have been implicated in the pathophysiology of female reproductive disorders. Nitroblue Tetrazolium (NBT) is a distinctive dye that assesses cellular redox activity in neutrophils through the detection of superoxide anion (O2-). However, the relationship between neutrophil-derived oxidative stress and women infertility remains to be fully elucidated. Aim of the study: We aimed to investigate the role of neutrophil oxidative stress in women infertility using a novel Nitroblue Tetrazolium (NBT) method developed in our laboratory. Materials and methodsBlood and serum specimens were obtained from a total of 100 Libyan women, comprising healthy fertile women (n = 21; controls) and infertile women (n = 79). Superoxide anion (O2-) generation in neutrophils was assessed using the novel NBT method, while malondialdehyde (MDA), a marker of lipid peroxidation, was determined using the thiobarbituric acid reactive substances (TBARS) assay. ResultsA significant increase in both NBT-reactivity levels and the percentage of NBT-positive neutrophils was observed in infertile women compared with healthy fertile controls (P < 0.0001). In addition, MDA levels were significantly higher in infertile women than in the control group, indicating enhanced lipid peroxidation. MDA levels were positively correlated with NBT-reactivity levels (r = 0.410, P = 0.0001) and the percentage of NBT-positive neutrophils (r = 0.21, P = 0.047). A significant positive correlation was also observed between NBT-reactivity levels and the percentage of NBT-positive neutrophils (r = 0.510, P = 0.0001), demonstrating a close association between neutrophil oxidative activity and lipid peroxidation in women infertility. ConclusionThe present study demonstrates the utility of a novel NBT method for detecting reactive oxygen species (ROS), a marker of oxidative stress, in neutrophils from both blood and serum specimens. The findings demonstrate increased neutrophil-derived oxidative stress in infertile women and suggest that this method may have potential as a diagnostic tool for the assessment of women infertility. ImpactThis study provides evidence that neutrophil-derived oxidative stress is significantly increased in women infertility and demonstrates the application of a novel Nitroblue Tetrazolium (NBT) assay for assessing oxidative stress in both blood and serum specimens. The significant associations between NBT-reactivity, NBT-positive neutrophils and malondialdehyde levels provide further insight into the contribution of oxidative stress to female reproductive pathophysiology. These findings support the potential utility of the novel NBT method as a simple and reliable diagnostic approach for evaluating oxidative stress in women infertility and provide a foundation for future studies investigating oxidative stress biomarkers in reproductive medicine.

Background: Early detection of preeclampsia with noninvasive and reliable biomarkers is the foremost step for minimizing adverse effects during pregnancy. However, none maternal serum analyte evaluated so far is sufficiently accurate to allow recommending their routine use. Methods: Microarray was used to first identify differentially expressed LncRNA and mRNA. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine bio-functions and signaling pathways. Quantitative real-time polymerase chain reaction was used to validate the results of microarray. Finally, the lncRNA-mRNA co-expression network was constructed to find the interaction of genes. Result: A total of 1476 differentially expressed lncRNAs and 594 mRNAs were identified. Both RNA-seq and RT-qPCR showed the expression of T241171,T338586, uc002ywy.3 was up-regulated, and the expression of ENST00000524858, T131416, T357032, uc.335+ was down regulated in the plasma of patients with preeclampsia. Pathway analysis showed the targeted mRNAs were enriched in apoptosis, sulfur metabolism, starch and sucrose metabolism. ceRNA network found that LncRNA-uc002ywy.3 might be the upstream regulator of miRNA-4498. LncRNA-uc002ywy.3/miRNA-4498 was predicted to interplay with genes involved in programmed cell death-1and its ligand (PD-1/PD-L1) pathway by KEGG analysis. Conclusion s PD-1/PD-L1 signaling pathway may be involved in the development of preeclampsia. The dysregulated LncRNA-uc002ywy.3/miRNA-4498 shed light on a new layer involved in the regulatory network of preeclampsia.

Objective Based on the bioinformatics method of multi-database integration, this study systematically analyzes the expression characteristics, clinical pathological correlation, prognostic value, potential molecular mechanisms, and immune infiltration patterns of hepatocyte growth factor receptor (MET) in gastric cancer. Methods The UALCAN and GEPIA databases were employed to examine the differential expression of MET between gastric cancer and normal gastric mucosal tissues, as well as its associations with clinicopathological features. Kaplan-Meier Plotter was utilized to evaluate the impact of MET expression on overall survival (OS) and progression-free survival (PFS). Protein-protein interaction (PPI) network was constructed via LinkedOmics, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-expressed genes. Four algorithms, TIMER, CIBERSORT, EPIC, and MCPcounter, were used to cross evaluate the correlation between MET expression and immune cell infiltration; Further validate the cell type specific expression of MET using the gastric cancer single-cell sequencing queues (GSE134520, GSE167297) built into the TISCH database. Results MET expression was significantly elevated in gastric cancer tissues compared with normal gastric mucosa (P < .05), and its expression level was significantly correlated with tumor grade and TNM stage. Patients with high MET expression exhibited significantly poorer OS and PFS than those with low MET expression (P < .05). The PPI network revealed that MET could interact with 20 key proteins, including EGFR, ERBB2, HGF, STAT3, and GRB2 etc. GO enrichment analysis suggests that differentially expressed genes are significantly enriched in functions such as the ERBB signaling pathway, cadherin binding, and DNA repair complexes; KEGG enrichment analysis showed that MET related genes were significantly enriched in pathways such as homologous recombination, nuclear cytoplasmic transport, mismatch repair, and oxidative phosphorylation. Immune infiltration analysis showed that the negative association between MET and B cells infiltration has cross algorithm robustness, while the association with neutrophils, CD8+ T cells, CD4+ T cells, and macrophages exhibits algorithmic heterogeneity or insignificance; There is no significant correlation between MET and common immune checkpoint molecules such as PD-1, PD-L1, CTLA4, etc. Single cell validation further confirmed that MET is mainly enriched in malignant epithelial cells and endothelial cells, and is almost not expressed in immune cells. Conclusions Multidimensional bioinformatic analyses demonstrate that elevated MET expression serves as an independent risk factor for unfavorable prognosis in gastric cancer. MET may mediate dual drug resistance in gastric cancer via crosstalk with multiple signaling molecules (including EGFR, ERBB2, HGF, STAT3 and GRB2) and dysregulation of the homologous recombination repair pathway. Results from multiple-algorithm immune infiltration analysis, single-cell dataset analysis and immune checkpoint correlation analysis indicate that MET exerts only modest direct regulatory effects on the gastric cancer immune microenvironment. This exploratory study offers systematic bioinformatic evidence supporting MET as a candidate prognostic biomarker and potential therapeutic target for gastric cancer. Further functional experiments and prospective cohort studies are required to validate its molecular mechanisms and clinical utility.

Breast cancer is a heterogeneous disease comprising molecular subtypes with distinct therapeutic vulnerabilities. Among emerging therapeutic strategies, copper-based nanoparticles have shown anticancer activity. However, whether these responses differ across breast cancer subtypes and which molecular programs underlie copper sensitivity remain poorly understood. We investigated subtype-specific responses to Polyvinylpyrrolidone-assisted copper nanoparticles (CuNP-PVP) in breast cancer cell lines and the molecular programs underlying copper adaptation. CuNP-PVP were synthesized and evaluated in luminal (MCF-7), HER2-positive (SKBR3), and triple-negative (MDA-MB-231) breast cancer cell lines, with transcriptomic analyses integrated with CCLE and TCGA-BRCA datasets. UV-Vis spectroscopy revealed a plasmon resonance band at 591 nm, while TEM showed spherical nanoparticles with an average diameter of 76.1 +/- 21.0 nm. DLS indicated a larger hydrodynamic diameter, consistent with PVP coating; {zeta}-potential measurements showed a surface charge of -13 mV, and XPS confirmed mixed reduced and Cu(I)/Cu(II) oxidation states. Exposure to 100 g/mL CuNP-PVP induced subtype-dependent effects on cell viability, with MCF-7 showing greater sensitivity, whereas MDA-MB-231 and SKBR3 were less affected. Baseline transcriptomic analyses revealed enrichment of oxidative stress, metabolic adaptation, and lysosomal programs in less sensitive cells, whereas MCF-7 showed higher expression of lipoylation-related genes. These features supported the development of a Potential Biological Copper-Response Index (PBCRI), which captured subtype-associated transcriptional patterns across independent cell lines and patient tumors, with luminal models showing lower scores and basal/TNBC models showing higher scores. Together, these findings identify candidate transcriptional programs associated with subtype-specific responses to CuNP and provide a framework for investigating potential molecular determinants of copper sensitivity.

Many factors including ionizing radiation and iodine deficiency are known to increase thyroid carcinogenesis risk. Our dataset analysis of The Cancer Genome Atlas (TCGA) showed that lower mRNA expression of NK2 homeobox 1 (NKX2-1) transcription factor, a master regulator of genesis, homeostasis, and function of thyroid, is linked to poor prognosis of papillary thyroid cancer patients. Here we provide the findings that thyroid-specific Nkx2-1 conditional knockout (Nkx2-1{Delta}T) mice develop thyroid adenoma and carcinoma in higher frequency with combined exposure to radiation and iodine deficiency than control Nkx2-1fl/fl mice. Iodine deficiency caused oxidative stress, which subsequently resulted in DNA damage, leading to transformation of thyroid follicular cells. RNA-seq gene set enrichment analysis indicated higher production of reactive oxygen species (ROS) in the thyroids of Nkx2-1{Delta}T as compared to Nkx2-1fl/fl mice with combined exposure to radiation and iodine deficiency. This was accompanied by a feedback induction of SOD3 (superoxide dismutase 3) and GPX2 (glutathione peroxidase 2). These antioxidants were naturally expressed at higher levels in the thyroids of Nkx2-1{Delta}T than Nkx2-1fl/fl mice without iodine deficiency or radiation. Nkx2-1{Delta}T thyroids exhibited abnormal follicle architecture and up-regulation of Acox2 (encoding acyl-CoA oxidase 2), which produces hydrogen peroxide. These results suggest that loss of NKX2-1 may contribute to excess ROS production, which elevates basal oxidative stress resulting in the promotion of ROS-induced carcinogenesis. We propose a role for NKX2-1 as a regulator of ROS production homeostasis in the thyroid. Its disturbance would dispose thyroid follicular cells more vulnerable to the ROS-producing carcinogens.

Background: Gastric carcinoma remains a major cause of cancer related mortality worldwide, with tumor progression increasingly recognized as a consequence of complex interactions within the tumor microenvironment. Hypoxia induced signaling, cancer associated fibroblast (CAF) heterogeneity, and immune checkpoint activation play critical roles in tumor progression and immune evasion. However, their integrated relationship in gastric carcinoma remains insufficiently characterized. Objectives: To evaluate the expression of Hypoxia inducible factor 1 alpha and its association with cancer-associated fibroblast subtypes and Programmed death-ligand 1 expression in gastric carcinoma. Methods: This cross sectional analytical study included 100 histologically confirmed gastric carcinoma cases from Satkhira Medical College. Immunohistochemistry was performed for HIF 1 alpha, smooth muscle actin (SMA), fibroblast activation protein (FAP), and PD L1. CAFs were subclassified into myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs). Associations between biomarkers and clinicopathological variables were analyzed using chi square test, Spearman correlation, and multivariate logistic regression. Receiver operating characteristic (ROC) curve analysis was used to assess model performance. Result: High HIF 1 alpha expression was observed in 55% of cases and demonstrated significant association with poor differentiation (p = 0.001), advanced tumor stage (p = 0.002), and lymph node metastasis (p = 0.001). iCAF predominance was significantly associated with poor differentiation (p = 0.003), advanced stage (p = 0.004), and nodal metastasis (p = 0.004). High PD L1 expression was significantly associated with poor differentiation (p = 0.03), advanced stage (p = 0.001), and lymph node metastasis (p = 0.002). Multivariate logistic regression identified high HIF 1 alpha expression (OR = 3.8, p = 0.001), iCAF dominance (OR = 4.5, p < 0.001), and advanced tumor stage (OR = 2.9, p = 0.004) as independent predictors of high PD L1 expression. Combined high HIF 1 alpha expression and CAF activation demonstrated the highest rate of PD L1 positivity (76.7%, p < 0.001). ROC curve analysis demonstrated good predictive performance of the model with an area under the curve of 0.81. Conclusion: The present study demonstrates a significant interaction between hypoxia, stromal remodeling, and immune checkpoint activation in gastric carcinoma. High HIF 1 alpha expression and inflammatory CAF predominance are strongly associated with aggressive clinicopathological features and increased PD L1 expression, supporting the existence of a coordinated hypoxia stroma immune axis in gastric carcinoma progression. These findings may have potential implications for prognostic stratification and combined targeted therapeutic strategies.

In marine teleosts, gill ionocytes play central roles in the excretion of excess monovalent ions (Na+, Cl-, and K+). Basolateral K+ channels are considered essential for sustaining ion secretion by maintaining K+ homeostasis and membrane potential; however, the molecular identity of basolateral K+ channels in teleost gill ionocytes remains incompletely understood. Kcnj15 is a K+ channel expressed in multiple osmoregulatory tissues of the Japanese pufferfish (Takifugu rubripes), and its localization changes depending on tissue type and osmotic conditions; however, its localization in the gills has remained unresolved. Here, we determined the cellular localization of Kcnj15 in the gills and examined its expression in response to changes in environmental salinity. In seawater-acclimated fish, Kcnj15 was localized to the basolateral membrane of Nka- and Nkcc1-positive ionocytes. Following acclimation to hypoosmotic brackish water (1 ppt), transcript levels of cftr, nkcc1, and kcnj1a decreased, whereas kcnj15 expression remained unchanged. Under this condition, Kcnj15 was localized to the basolateral membrane of Nka-positive ionocytes. These results demonstrate that Kcnj15 is localized to the basolateral membrane of gill ionocytes and suggest that it may function as a basolateral K+ leak channel supporting ionocyte homeostasis under both seawater and low-salinity conditions.

Valproic acid/sodium valproate (VPA) is a widely prescribed anticonvulsant and has also been used against certain tumor cells. It is a potent modulator of gene expression. Its ability to induce apoptosis has been well documented in HeLa cells. However, another form of cell death - mitotic catastrophe - has not yet been explored in VPA-treated HeLa cells. Here, we investigated the effects of VPA treatment on mitotic catastrophe characteristics, including morphological features and their frequencies, fluorescence intensity signals of caspase-2 and p53, and the expression and abundance of DNMT1 and DNMT3B. An increased frequency of mitotic catastrophe was observed not only morphologically, but also through enhanced induction of caspase-2, involvement of p53, at least under more drastic VPA treatment, but without a decrease in DNMT1 or DNMT3B levels. Additionally, enhancement of mitotic catastrophe coincided with a reduction in mitotic chromosome abnormalities. Increased DNMT3B expression following VPA action, may be favored by previously reported chromatin decondensation induced by this drug. Enhanced CpG methylation of specific DNA sites could thus be promoted. In conclusion, VPA was shown to trigger metabolic pathways linked to different forms of cell death in HeLa cells, supporting its oncosuppressive potential.

BackgroundAnesthetic agents administered during surgery are one of the key perioperative factors affecting immune modulation in cancer patients. This comparative study elucidated the mechanisms by which the volatile anesthetic, isoflurane and the intravenous anesthetic, propofol impacted apoptosis signaling in CD4+ helper T (Th) cells. MethodsFlow cytometry was used to analyze apoptosis, mitochondrial function and reactive oxygen species (ROS) generation, while Western blotting, ELISA and RT-qPCR were employed to study protein/gene expression in sorted CD4{square} Th cells from perioperative breast cancer female patients (anesthetized with isoflurane or propofol, n=15 per group) and Jurkat T cells. ResultsPatient-derived CD4{square} Th cells and Jurkat T cells exhibited that isoflurane at clinically relevant concentrations triggered apoptosis through mitochondrial depolarization, ROS generation, DNA damage, and activation of caspase-3/7. Specific use of caspase-3/7 inhibitor, Z-DEVD-FMK and antioxidant N-acetyl cysteine rescued isoflurane-induced apoptosis. Further, isoflurane relative to propofol, activated p38 mitogen-activated protein kinase (MAPK), and use of p38 inhibitor, SB203580 suppressed isoflurane-induced apoptosis. Collectively, these findings validated the involvement of the ROS-p38-caspase-3/7 axis in isoflurane-associated apoptosis signaling. On the other hand, propofol conserved mitochondrial integrity, reduced oxidative stress, and maintained higher proliferative capacity. Interestingly, isoflurane-associated apoptosis was transient, with postoperative recovery in patients and similar rescue from apoptosis was evident in Jurkat T cells within 24-48 h of drug removal. ConclusionsBy integrating analyses of patient-derived CD4+ Th cells with mechanistic validations in Jurkat T cells, this study identified the ROS-p38-caspase-3/7 signaling axis and the reversible nature of isoflurane-induced apoptosis.

Ovarian cancer stands out as one of the most lethal gynecological cancers among women in the United States. While patients with ovarian cancer often show positive responses to treatment, it is common for the cancer to reappear and develop resistance to treatment, so it is important to explore and create novel combinations to increase patient survival rates. ATRi (Ataxia telangiectasia and Rad3-related inhibitors) and ONC201 monotherapies have shown efficacy in ovarian cancer in preclinical models. We hypothesized that combining ATRi with ONC201 would enhance this efficacy. ONC201 induces apoptosis through the TRAIL-pathway following activation of the integrated stress response and inactivation of Akt which is upregulated in ovarian cancer. Six human ovarian cancer cell lines were treated alone and with the novel drug combination of ceralasertib (ATRi) and ONC201. We found an IC50 range of 0.88-20.16 microM for ONC201 and IC50 range of 0.55-54.75 microM for ceralasertib. The combination of ONC201 plus ceralasertib resulted in PARP cleavage consistent with apoptosis along with reduction in Bcl-XL, Xiap and p-Akt expression after 72 hours of treatment. Cytokine profiling showed a decrease in markers of inflammation and tumor progression. Our data provide evidence of synergy from the combination of ATRi ceralasertib and ONC201 against Ovarian cancer cells that merits further investigation including clinical translation.

Melittin exhibits antitumor activity in cervical cancer models, yet the long non-coding RNA (lncRNA) response and associated regulatory networks remain poorly understood. Here, strand-specific RNA-seq data from melittin-treated and untreated U14 murine cervical cancer cells were analyzed to characterize melittin-responsive lncRNAs and explore their potential functional associations. A total of 28,162 lncRNAs were identified, including 27,307 known and 855 novel transcripts. Differential expression analysis revealed 404 differentially expressed lncRNAs (DElncRNAs), comprising 191 upregulated and 213 downregulated lncRNAs, w most of which were predicted to localize to the cytoplasm or nucleus. Cis-target analysis identified 52 neighboring mRNAs as putative targets of 46 DElncRNAs. Functional enrichment highlighted mitochondrial electron transfer and redox-related processes, including the mitochondrial electron transfer flavoprotein complex, electron-transferring-flavoprotein dehydrogenase activity, ubiquinone binding, and quinone binding. In parallel, melittin induced mitochondrial membrane depolarization and increased intracellular reactive oxygen species accumulation in U14 cells. Co-expression analysis further identified 138 lncRNAs co-expressed with 161 mRNAs, which were enriched in chromatin remodeling, DNA replication, and DNA repair. EdU incorporation decreased with increasing melittin concentrations, indicating suppression of DNA synthesis and proliferative activity. RT-qPCR analysis confirmed the expression trends of selected DElncRNAs. Collectively, these findings demonstrate extensive remodeling of the lncRNA landscape in melittin-treated U14 cells and suggest that melittin-responsive lncRNA-mRNA networks are associated with mitochondrial redox disruption and impaired DNA synthesis. This study provides a transcriptomic framework for identifying candidate lncRNA-mRNA regulatory axes underlying the antitumor response to melittin.

Heavy metal contamination of aquatic environments poses persistent risks to ecosystems and human health. Aquatic macrophytes such as Pistia stratiotes are increasingly used for phytoremediation due to their capacity to accumulate contaminants without displaying symptoms of abiotic stress. However, the molecular mechanisms underlying their metal tolerance remain poorly understood. Metallothioneins (MTs) are cysteine-rich proteins involved in metal binding and detoxification in plants. In this study, genome-wide analysis was performed to identify and characterise candidate MT genes in P. stratiotes and to assess their transcriptional response to copper exposure. Six novel MT genes (PsMT2f, PsMT3a-d, and PsMT4) were identified and classified into MT2, MT3, and MT4 subfamilies based on conserved cysteine motifs and domain architecture. Quantitative real-time PCR showed differential and tissue-specific expression of MT genes in roots and leaves following exposure to copper for up to 4 h. These findings provide the first comprehensive characterisation of the MT gene family in P. stratiotes and demonstrate their potential role in copper tolerance, supporting the application of this species in the phytoremediation of metal-contaminated aquatic systems.