Search Results (988)

Background: Hepatocellular carcinoma (HCC) remains a major clinical challenge due to its pronounced molecular heterogeneity and frequent resistance to conventional therapies. A key driver of therapeutic failure is the overexpression of the anti-apoptotic proteins myeloid cell leukemia-1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-XL), which collectively maintain mitochondrial integrity and promote tumor cell survival. Methods: In this study, we evaluated a rational combination strategy targeting these complementary survival pathways using the histone deacetylase inhibitor panobinostat and the DNA-damaging agent bleomycin in HepG2 cells, a p53-functional HCC cell model. Results: In HepG2 cells, each agent alone produced only limited cytotoxicity, whereas their combination resulted in a marked and synergistic induction of apoptosis. This was shown by increased Annexin V positivity, mitochondrial outer membrane permeabilization (MOMP), and activation of caspases-8, -9, and -3 as well as cleavage of poly(ADP-ribose) polymerase (PARP). Mechanistically, panobinostat reduced Bcl-XL expression and primed mitochondria for apoptosis but simultaneously triggered compensatory upregulation of Mcl-1, representing an adaptive resistance response within this experimental system. Bleomycin effectively counteracted this escape mechanism by suppressing Mcl-1 induction, thereby lowering the apoptotic threshold and enabling mitochondrial permeabilization. In parallel, combined treatment potentiated caspase-8 cleavage, suggesting an additional caspase-8-associated apoptotic signal that amplified caspase-3/PARP execution. Pharmacological inhibition with zVAD-FMK confirmed that the observed cell death was predominantly caspase-dependent, supporting a coordinated engagement of both intrinsic and extrinsic apoptotic pathways. In summary, the combination of panobinostat and bleomycin overcomes anti-apoptotic defenses in HepG2 cells through synergistic and coordinated disruption of mitochondrial survival checkpoints and dual apoptosis pathway activation. Conclusions: By blocking a compensatory Mcl-1 escape response while simultaneously engaging extrinsic apoptosis signaling, this strategy produces potent synergistic cell death in this defined p53-functional HCC model and represents a promising mechanistic proof of concept that warrants further validation in additional molecularly diverse HCC models before broader translational conclusions can be drawn. Full article

The calcium ion-transporting ATPase (Ca²⁺-ATPase) gene family maintains plant intracellular Ca²⁺ homeostasis and regulates growth, development and stress immunity; however, its functions remain poorly characterized in Malus domestica. Here, we performed a genome-wide identification of apple Ca²⁺-ATPase genes and obtained 45 members, which were classified into MdACA (39) and MdECA (6) subfamilies and unevenly distributed on 14 chromosomes. Phylogenetic analysis of Ca²⁺-ATPase genes from Malus domestica, Arabidopsis thaliana, and Oryza sativa classified these proteins into five subgroups. The ACA and ECA subfamilies were highly conserved across species, whereas Group D was apple-specific. Collinearity and Ka/Ks analyses indicated that segmental duplication and purifying selection dominated the evolution of apple Ca²⁺-ATPase genes. Promoter cis-element prediction uncovered numerous regulatory elements related to phytohormone signaling, growth, development and stress defense. Codon usage bias analysis indicated that AUG (methionine) was the dominant codon. Tissue expression profiles showed differential expression of apple Ca²⁺-ATPase genes in various organs. Quantitative real-time PCR (qRT-PCR) assays demonstrated widespread responses of Ca²⁺-ATPase genes to Alternaria alternata infection, exogenous CaCl₂, salicylic acid (SA) and methyl jasmonate (MeJA), among which MdACA39 was strongly induced under all treatments. Subcellular localization verified that MdACA39 resides on the plasma membrane. Moreover, transient overexpression of MdACA39 significantly enhanced apple resistance to A. alternata, likely due to the activation of SA, MeJA and Ca²⁺ signaling-mediated immune pathways, the induction of disease resistance-related genes, and elevated antioxidant enzyme activity. Collectively, this study systematically characterizes the apple Ca²⁺-ATPase family and identifies MdACA39 as a key regulator of fungal resistance, providing valuable gene resources for dissecting Ca²⁺ signaling-mediated disease resistance in apple. Full article

Redox regulation plays an important role in plant stress responses. Our previous study revealed that rice GLUTATHIONE PEROXIDASE 1 (GPX1) acts as a redox sensor and transducer and promotes osmotic stress tolerance by transfer of cytosolic oxidative signals to transcription factor BASIC LEUCINE ZIPPER 68 (bZIP68). However, the mechanisms governing GPX1 activity and nuclear localization remain unclear. Here, we show that osmotic stress increases GPX1 acetylation. Peroxidase activity and subcellular localization assay indicated that the effects of acetylation on GPX1 function are site-specific, as the acetylation of K94 and K121 enhances GPX1 enzymatic activity, whereas the C-terminal K159/K162/K163 cluster is required for its nuclear translocation. Transgenic complementation and physiological assays confirmed that substitution of K159/K162/K163 sites into arginine abolished GPX1-mediated osmotic stress tolerance and the activation of bZIP68 target genes. Furthermore, we discovered that HISTONE DEACETYLASE 15 (HDA15) interacts with and deacetylates GPX1. HDA15-mediated deacetylation reduced enzymatic activity, nuclear translocation and subsequently the interaction with bZIP68 of GPX1. Accordingly, HDA15-overexpressing rice showed greater membrane damage, weaker induction of bZIP68-regulated genes and increased sensitivity to osmotic stress. These results identify HDA15-mediated GPX1 deacetylation as a negative regulatory mechanism that connects redox enzyme activity, protein localization and stress-responsive transcription in rice. Full article

Cadmium (Cd) is a toxic metal that poses a significant threat to crop production and global food security. Transporters play a critical role in mediating the uptake of metal ions, including Cd. However, a substantial number of Cd transporters in rice remain uncharacterized. In this study, we identify OsMDR4, a member of the multidrug resistance protein family, as a mediator of Cd uptake in rice. Heterologous overexpression of OsMDR4 in yeast increased both Cd sensitivity and intracellular Cd accumulation. Consistent with this, the Cd concentrations in both roots and shoots of the mdr4 mutants were significantly lower than those in wild type. Kinetic analysis further revealed that the maximum Cd uptake rate in mdr4 mutants was markedly reduced compared with wild type. Expression analysis showed that OsMDR4 is primarily expressed in the epidermis and root hairs of rice seedlings and in floral organs during the flowering stage. Notably, OsMDR4 expression in seedling roots was upregulated in response to Cd exposure. Subcellular localization analysis revealed that OsMDR4–EGFP was predominantly localized to the plasma membrane in a heterologous Arabidopsis protoplast system. In summary, we have identified and characterized OsMDR4 as a previously uncharacterized protein that contributes to cadmium accumulation in rice. Full article

Inorganic polyphosphate (polyP) is a ubiquitous polymer increasingly recognized as a regulator of protein function through lysine polyphosphate modification (KPM), a reversible interaction with lysine-rich protein regions. Although numerous KPM targets have been identified, the functional consequences of polyP binding remain poorly understood. N-myristoyltransferase 2 (NMT2), an essential enzyme that catalyzes protein N-myristoylation and regulates membrane-associated signaling pathways, was previously identified as a candidate KPM target. Here, we investigated the molecular and functional relationship between polyP and NMT2. Using a fluorescence-based coenzyme A release assay, we found that polyP directly down-regulated NMT2 enzymatic activity in a dose-dependent manner, with long-chain polyP (polyP₇₀₀) producing significantly greater inhibition than medium-chain polyP (polyP₁₀₀). In cells, both exogenous polyP treatment and induction of endogenous polyP synthesis reduced phosphorylation of Src, a downstream signaling protein whose activation depends on N-myristoylation, supporting inhibition of NMT2 function in vivo. PolyP also decreased the viability of NMT2-overexpressing HeLa cells in a dose-dependent manner, suggesting functional consequences for cellular fitness. Biochemical analysis further revealed that polyP induced a pronounced electrophoretic mobility shift in NMT2 that was completely reversed by a 25-residue lysine-rich competitor peptide, consistent with a specific and reversible interaction mediated through lysine-enriched regions of the enzyme. These findings identify NMT2 as a functional target of polyP and demonstrate that polyP negatively regulates NMT2 activity, downstream Src signaling, and cellular viability. Our results expand the emerging concept of lysine polyphosphate modification and establish polyP as a previously unrecognized regulator of protein lipidation-dependent signaling pathways. Full article

To preliminarily elucidate the bidirectional metabolic effects caused by changes in PFKM expression, provide research clues for further investigation of the molecular mechanisms through which PFKM regulates porcine skeletal muscle metabolism and myogenic differentiation, and offer a reference for identifying candidate genes associated with meat production traits and studying gene regulation in pig breeding, this study compared skeletal muscle protein expression profiles between Large White and Bama pigs. Candidate regulatory factors related to muscle growth and energy metabolism were screened, and the effects of altered muscle-type phosphofructokinase (PFKM) expression on metabolic homeostasis and myogenic differentiation in porcine skeletal muscle satellite cells (SMSCs) were preliminarily evaluated. Longissimus dorsi muscle tissues from Large White and Bama pigs were analyzed using iTRAQ-based proteomics. A total of 2040 reliably quantified proteins were identified, of which 51 were relatively upregulated in Large White pigs and 73 were relatively upregulated in Bama pigs. Functional enrichment analysis showed that the differentially expressed proteins were mainly involved in glycolysis, mitochondrial energy metabolism, protein synthesis, and the regulation of muscle fiber structure and function. PFKM was therefore selected as a key candidate differentially expressed protein. Porcine SMSC models comprising a PFKM knockdown group (PFKM-KD), a PFKM overexpression group (PFKM-OE), and a normal control group (PFKM-CON) were subsequently established. Glucose consumption and lactate accumulation in the culture medium, ATP levels, reactive oxygen species (ROS), mitochondrial membrane potential, apoptosis, mitochondrial dynamics-related proteins, and myogenic differentiation markers were then examined. Compared with the PFKM-CON group, the PFKM-OE group showed significantly increased glucose consumption and lactate accumulation, together with significant increases in ROS levels, mitochondrial membrane potential, and apoptosis, whereas ATP levels were significantly reduced. In the PFKM-KD group, glucose consumption and lactate accumulation were significantly decreased, accompanied by reductions in mitochondrial membrane potential, ROS, ATP levels, and apoptosis. PFKM overexpression mainly induced oxidative stress, ATP depletion, and increased apoptosis, whereas PFKM knockdown primarily reduced mitochondrial membrane potential, ROS, and ATP levels, indicating a relatively low-metabolic state. Both treatments were accompanied by dysregulated expression of the mitochondrial dynamics-related proteins DRP1, MFN2, and OPA1, although their patterns of change were not identical. Western blotting and immunofluorescence consistently showed that the expression levels of the myogenic differentiation markers MyoD and MYH were significantly lower in both the PFKM-KD and PFKM-OE groups than in the control group, suggesting that either excessive or insufficient PFKM expression may impair the myogenic differentiation potential of SMSCs. In conclusion, changes in PFKM expression are closely associated with glycolysis-related metabolism, energy and redox homeostasis, mitochondrial function-related indicators, and myogenic differentiation capacity in porcine SMSCs. The normal biological function of PFKM may therefore depend on its expression being maintained within an appropriate range. Full article

Glioblastoma (GBM) is a devastating brain tumor heavily reliant on metabolic reprogramming for survival. The DNA aptamer AS1411 specifically targets cell-surface nucleolin (NCL), a protein overexpressed in GBM; however, its precise metabolic consequences remain largely unexplored. This study investigated the direct impact of AS1411-mediated NCL inhibition on the lipidomic profile of U-87 glioblastoma cells. It was demonstrated that AS1411 treatment induced acute cytotoxicity within 24 h. Subsequently, high-resolution mass spectrometry (MS) lipidomics was utilized to identify the lipidomic rewiring triggered by AS1411. Significant changes, such as the upregulation and/or exclusive emergence of specific long-chain and highly polyunsaturated diacylglycerol (DAG), triacylglycerol (TAG), and glycerophospholipid (GP) species, were determined in the species-level analyses. Additionally, our findings revealed that AS1411 treatment induced substantial alterations that profoundly affected membrane biophysics by modifying lipid saturation and acyl chain lengths. An increase in fully saturated sphingomyelin (SM) and cholesteryl ester (CE) levels was observed, leading to the formation of saturated lipid microdomains (lipid rafts) in endosomal and ER membranes, which causes membrane rigidification and decreased fluidity. Our results also demonstrate that PEs containing long-chain polyunsaturated fatty acids (PUFAs)—the primary substrates for ferroptosis—were upregulated. While AS1411 subjects cancer cells to methuotic vacuolization stress, it simultaneously reduces internal structural membrane fluidity and renders the cells metabolically vulnerable to ferroptosis. In conclusion, these detailed lipidomic results indicate that AS1411 treatment proceeds strictly through targeted remodeling and an adaptive scaffolding response. Full article

Maize (Zea mays L.) productivity is severely constrained by drought stress. Although the maize SET domain-containing gene family has previously been investigated, the earlier analysis was based on the incomplete B73 RefGen_v2 genome assembly, and the drought-responsive functions of individual ZmSET members remain largely uncharacterized. In this study, 47 ZmSET genes were identified using the updated B73 RefGen_v5 genome and systematically analyzed for their physicochemical properties, chromosomal distribution, gene structures, conserved motifs, and promoter cis-acting elements. The ZmSET family exhibited substantial evolutionary conservation, while its promoters contained numerous stress- and hormone-responsive elements. Transcriptome analysis identified ZmSET9 as a drought-responsive gene, and RT-qPCR showed that it maintained relatively high expression throughout drought treatment. Heterologous overexpression of ZmSET9 in Arabidopsis thaliana enhanced drought tolerance and supported plant growth under drought stress. Compared with wild type plants, the transgenic lines exhibited higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities and lower malondialdehyde (MDA) contents, indicating enhanced antioxidant capacity and reduced membrane lipid peroxidation. Under PEG induced osmotic stress, AtCAT1 and AtMYC2 were more strongly induced in the transgenic lines. Protein–protein interaction prediction and yeast two-hybrid assays further demonstrated that ZmSET9 physically interacts with FERTILIZATION-INDEPENDENT ENDOSPERM 1 (FIE1), a core component of Polycomb repressive complex 2. These findings update the genomic characterization of the maize SET family and suggest that ZmSET9 contributes to drought tolerance by enhancing antioxidant defense and regulating stress-responsive gene expression. Full article

Heat shock proteins (HSPs), particularly HSP70 and HSP90, are highly conserved molecular chaperones that protect cells from a wide range of stressors and maintain proteome homeostasis. In cancer, tumor cells frequently overexpress and actively release HSPs into the extracellular space and circulation in response to metabolic alterations, hypoxia, oxidative stress, and therapeutic interventions. Consequently, circulating HSP levels are often elevated in patients with malignancies compared with healthy individuals. This review summarizes current evidence on the diagnostic, prognostic, and predictive value of circulating HSPs in both solid and hematological cancers. Clinical studies indicate that circulating HSP concentrations are associated with tumor type, disease stage, lymph node involvement, metastatic burden, treatment response, and risk of recurrence. Importantly, membrane-associated and extracellular vesicle-associated forms of HSP70 appear to exhibit greater tumor specificity than freely circulating proteins, highlighting the importance of selecting appropriate analytical approaches for biomarker assessment. Beyond their utility as biomarkers, extracellular HSPs actively participate in tumor biology and anti-tumor immunity. Depending on the cellular and immunological context, they can either support tumor progression or stimulate immune responses through activation of natural killer cells, antigen-presenting cells, and cross-presentation of tumor-derived antigens. These immunomodulatory properties have provided the foundation for the development of HSP-based vaccines and adoptive immunotherapeutic strategies, several of which have demonstrated encouraging results in clinical trials. We further discuss the relationship between extracellular chaperone biology and responses to major anticancer treatments, including radiotherapy, chemotherapy, HSP90-targeted therapies, and immune checkpoint blockade. In conclusion, the available evidence supports circulating extracellular HSPs as promising non-invasive biomarkers and potential pharmacodynamic indicators that may improve patient stratification, treatment monitoring, and prediction of therapeutic efficacy in clinical oncology. Full article

Myeloid cell leukemia-1 (MCL-1) is a cellular survival protein belonging to the Bcl-2 protein family and is overexpressed in human colorectal cancer (CRC). Background: This study aimed to reduce cancer progression by suppressing the MCL-1 protein using PROTAC MCL-1 Degrader-1 and Trametinib, with the aim of overcoming the apoptosis resistance caused by high MCL-1 expression levels in colorectal cancer cells. Methods: Therefore, we tested the cell viability, proliferation, mitochondrial membrane potential, cell cycle progression, and cell death potential of MCL1-specific PROTAC Mcl-1 Degrader-1 and the combination of PROTAC Mcl-1 Degrader-1 and Trametinib in colorectal cancer cell lines using different methods such as WST-8 assays, real-time cell analysis, MMP/JC-1 staining assays, flow cytometry, and Western blot analysis. Results: The results suggest that PROTAC Mcl-1 Degrader-1 is associated with dose- and time-dependent reductions in cell proliferation in colorectal cancer cells. Under the experimental conditions used in this study, PROTAC MCL-1 Degrader-1 showed limited effects on the migratory potential of colorectal cancer cells but was associated with changes in cell cycle distribution, including an increased proportion of HT-29 cells in the G2/M phase. In addition, combination treatment with PROTAC MCL-1 Degrader-1 and trametinib was associated with greater reductions in cell viability and proliferation than either monotherapy. The combination treatment was also accompanied by changes in mitochondrial membrane potential and increased apoptotic cell populations, with a higher proportion of early apoptotic cells observed in HT-29 and COLO-205 colorectal cancer cell lines. Conclusion: Our findings suggest that the reduction in MCL-1 protein levels observed following PROTAC Mcl-1 Degrader-1 treatment may contribute to its potential therapeutic relevance in colorectal cancer. Full article

Background: Multidrug efflux proteins, frequently overexpressed in cancer cells, reduce intracellular drug accumulation and limit chemotherapeutic efficacy. Short drug-binding peptides containing the WXXW motif have been shown to non-covalently bind multidrug resistance (MDR)-associated drugs, potentially masking structural features recognized by efflux transporters. We hypothesized that incorporating this motif into a membrane-active peptide would generate a hybrid analogue capable of reversible drug binding and enhanced cellular uptake. Methods: A melittin-derived peptide (M3) was rationally designed by introducing the WXXW motif into the flexible region adjacent to the conserved proline kink to generate a dual-functional peptide with membrane activity and reversible drug binding. Membrane activity was evaluated using liposome leakage assays, and drug-binding interactions with doxorubicin were characterized using fluorescence quenching and microscale thermophoresis (MST). Molecular dynamics simulations were performed to elucidate binding mechanisms, and functional effects were assessed using calcein AM efflux assays, confocal imaging, and cytotoxicity studies across cancer cell lines. Results: M3 retained membrane activity and exhibited moderate, reversible binding to doxorubicin, with simulations showing binding initiation at the WXXW motif and extension to tryptophan residues W12, W15, and W19, forming a multivalent aromatic interface that suggested shielding of key drug functionalities. Functionally, M3 enhanced intracellular calcein retention and increased doxorubicin accumulation, and combination treatment produced synergistic cytotoxicity in the multidrug-resistant H69AR cell line with reduced toxicity toward normal epithelial cells. Conclusions: M3 acts as a membrane-active, reversible drug-binding peptide that enhances intracellular drug accumulation, supporting its potential as a modular strategy to overcome efflux-mediated MDR. Full article

Background: Tight junctions are major components of apical junction complexes and are crucial for the maintenance of cell polarity, healthy tissue architecture, adhesion, and permeability. These junctions include the claudin family of transmembrane proteins, which act as paracellular barriers to regulate selective permeability. Abnormal claudin expression disturbs cell adhesions and is associated with cancer through promoting cell invasion, migration, and metastasis. Claudin 6 (CLDN6) overexpression, in particular, is linked to several types of cancer with malignant phenotypes. The present study aimed to investigate the association between CLDN6 protein expression and its copy number variations (CNVs) with clinicopathological features and survival outcomes of ovarian cancer (OC) patients. Methods: A total of 114 formalin-fixed paraffin-embedded blocks from primary OC patients were used to construct tissue microarray slides. Automated immunostaining was used to assess CLDN6 protein expression levels, and next-generation knowledge discovery platforms were used to further evaluate CLDN6 CNV levels using The Cancer Genome Atlas open-source data. The relationships between CLDN6 CNVs and tumor stage, overall survival, disease-specific survival (DSS), and disease-free survival (DFS) were investigated. Results: This study demonstrated that CLDN6 had a mixed membranous-cytoplasmic expression pattern. The cytoplasmic expression of CLDN6 was significantly associated with tumor stage (p = 0.05), tumor size (p = 0.04), and recurrence (p = 0.05). In Univariate analysis, Kaplan–Meier analysis demonstrated that CLDN6 expression was significantly correlated with DFS (p = 0.01). OC patients with lower cytoplasmic CLDN6 expression levels lived longer and had lower recurrence rates. These findings were further confirmed through CLDN6 CNVs analysis, where OC with lower CLDN6 cytoplasmic expression positively correlated with longer DFS and DSS. No independent prognosticator was found when using Cox-regression multivariate analysis (p > 0.05). Conclusions: These results suggest CLDN6 as an interesting prognosticator to identify OC patients at a higher risk of recurrence in order to provide personalized management, alleviate the burden of this disease on women’s health, and improve their survival outcomes. Full article

The non-specific lipid transfer proteins (nsLTPs) are able to bind various hydrophobic compounds and facilitate the transport of fatty acids between intracellular membranes, and nsLTPs are found in rice endosperm and embryo during seed development. However, whether nsLTPs function as lipid carriers and thereby affect lipid metabolism in rice grains remains unclear. To elucidate whether nsLTPs influence fatty acid distribution in rice, we generated OsLTP10-OE (OsLTP10 overexpression) and OsLTP10-CR (OsLTP10 CRISPR/Cas9) lines. Phenotypic and metabolic analyses indicated that OsLTP10 expression is closely associated with fatty acid (FA) profiles and grain appearance. In general, total fatty acid content in the brown rice of OsLTP10-OE was higher than that in wildtype, but OsLTP10-CR was lower than wildtype. While FA accumulation was altered in both tissues, the endosperm (milled grain) was more severely affected than the bran, with individual FAs in the milled grains of OsLTP10-OE expanding by 31.87–52.00%. Additionally, key grain quality traits were substantially altered; OsLTP10-CR lines displayed a significantly enlarged white-belly chalkiness area alongside a 19.50% reduction in amylose content, whereas OsLTP10-OE lines showed decreased chalkiness and a 7.80% increase in amylose. Overall, the fatty acid content and composition, chalkiness, brown rice size, and amylose were influenced by OsLTP10. Full article

Reticulon-like proteins shape the endoplasmic reticulum (ER) membrane network, yet the developmental and physiological roles of individual plant reticulon isoforms remain poorly understood. Here, we characterize an Arabidopsis RTNLB16 T-DNA allele, rtnlb16-1, that exhibits severe photoperiod-dependent growth retardation and chlorosis. Molecular analysis revealed that rtnlb16-1 is not a simple loss-of-function mutant: the T-DNA insertion deletes the 5′ region required for RTNLB16 splice variant 7, while a CaMV35S enhancer associated with the insertion drives overexpression of the remaining splice variants. This misexpression is enhanced under long-day photoperiods and reduced under continuous low light, paralleling the severity of the mutant phenotype and its partial rescue. RTNLB16.5-GFP localized mainly to the tubular ER network and punctate cell-boundary structures consistent with plasmodesmata-associated ER. Neither overexpression of RTNLB16 isoforms 1–6 nor CRISPR-Cas9 disruption of major RTNLB16 isoforms reproduced the rtnlb16-1 phenotype, supporting a model in which altered splice-variant stoichiometry, rather than simple loss or gain of function, underlies the developmental defects. Transcriptome profiling showed that rtnlb16-1 undergoes extensive photoperiod-dependent transcriptional reprogramming, including changes in defense, hormone-response, senescence, photosynthesis, and iron/redox-associated gene networks. Physiologically, rtnlb16-1 displayed enhanced recovery from dark-induced senescence, while both rtnlb16-1 and rtnlb16-2 showed reduced sensitivity to exogenous abscisic acid during germination. Together, these findings suggest that balanced expression of RTNLB16 splice variants is important for normal growth and for coordinating ER-associated stress, hormone, and senescence responses in Arabidopsis. Full article

Glutamate excitotoxicity is a critical pathological mechanism underlying neuronal death in ischemic stroke, epilepsy, and neurodegenerative diseases. Caveolin-1 (Cav-1), a structural protein of caveolae membrane microdomains, has emerged as a potential modulator of neuronal survival, yet its precise mechanisms in excitotoxicity remain incompletely understood. In this study, we investigated the role of Cav-1 in regulating glutamate-induced calcium dysregulation, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in primary hippocampal neurons. Using Cav-1 overexpression (Cav-1OE) and Cav-1 knockdown (Cav-1KD) approaches, we demonstrate that Cav-1OE significantly attenuates glutamate-stimulated intracellular Ca²⁺ elevation, reduces ROS generation, and prevents mitochondrial membrane potential (Ψ_m) depolarization. Further investigation revealed that Cav-1OE reduces, while Cav-1KD enhances, calcium responses mediated by NMDA, AMPA, and KA receptors. These findings establish that Cav-1 functionally attenuates excitotoxic signaling by negatively regulating ionotropic glutamate receptor-mediated Ca²⁺ influx. Full article