Search Results (117)

Melanoma is a highly aggressive malignancy characterized by strong invasive and metastatic potential. CPEB4 has been implicated in melanoma progression and may serve as a potential therapeutic target. Alkannin has previously been reported to exert antitumor activity against melanoma; however, its in vivo efficacy and direct molecular interaction with CPEB4 remain unclear. In this study, a subcutaneous xenograft model using BALB/c nude mice was used to assess the in vivo antitumor effects of alkannin, and CPEB4 expression was analyzed via Western blotting. DARTS, CETSA, and SPR investigations were used to elucidate the interaction between alkannin and CPEB4. In addition, stable CPEB4-knockdown A375 melanoma cells were established to examine the effects of alkannin on cell proliferation, apoptosis, cell cycle progression, migration, invasion, and downstream signaling molecules. Alkannin markedly suppressed tumor growth in the xenograft model and reduced CPEB4 expression in a dose-dependent manner compared with the model group. DARTS and CETSA demonstrated alkannin-induced stabilization of CPEB4, while SPR analysis using purified recombinant CPEB4 showed a direct physical interaction with alkannin, with micromolar affinity. At the molecular level, alkannin downregulated CPEB4 and PRC1 expression (p < 0.05), whereas CPEB4 knockdown markedly suppressed MITF and PRC1 (p < 0.05). Notably, alkannin treatment alone did not significantly alter MITF protein expression under the present experimental conditions. Alkannin exerts antitumor activity against melanoma, while its interaction with CPEB4 and the associated molecular changes may contribute to cellular responses involving proliferation, survival, migration, invasion-related phenotypes, and mitotic regulation. Full article

Background and Objectives: Sevoflurane, a widely used inhalational anesthetic, is frequently administered during hepatocellular carcinoma (HCC) surgery, including hepatic resection and orthotopic liver transplantation. Because such procedures often require prolonged anesthetic exposure, the potential influence of sevoflurane on HCC cell behavior is of clinical interest. We aimed to evaluate the effects of sevoflurane on the proliferation and migration of HepG2 cells in vitro and on tumor growth in a xenograft mouse model in vivo, and to explore whether hypoxia-inducible factor-1α (HIF-1α) might be involved in this process. Materials and Methods: For the in vitro experiments, HepG2 cells were exposed to room air (0%), 2%, or 4% sevoflurane. A scratch wound healing assay was used to assess cell migration, and the number of viable cells was quantified by hemocytometer counting on day 4 to estimate proliferation. For the in vivo experiments, BALB/c nude mice bearing HepG2 xenografts were exposed to room air, 2% sevoflurane, or 4% sevoflurane for 3 h, three times weekly for 5 weeks. Tumor size and tumor weight were measured at the end of the exposure period. HIF-1α protein levels in tumor tissue were measured by enzyme-linked immunosorbent assay (ELISA) in tumor lysates and normalized to total tumor protein as an exploratory mechanistic analysis. Given the small sample available for this endpoint, the analysis had limited sensitivity to detect modest differences. Results: When wound closure was quantified and pooled across the analyzable experiments, no statistically significant difference was detected among the room air, 2% sevoflurane, and 4% sevoflurane groups (day-2 closure 19.9 ± 32.1%, 22.1 ± 25.8%, and 22.3 ± 28.8%, respectively; repeated-measures ANOVA p = 0.82), with variability dominated by between-experiment rather than treatment differences. In the proliferation assay, the number of viable HepG2 cells on day 4 was significantly lower in the 2% sevoflurane group (62.6 ± 3.3 × 10⁵) than in the room air group (68.5 ± 4.2 × 10⁵; p < 0.05); the 4% sevoflurane group (66.0 ± 3.2 × 10⁵) showed an intermediate value that did not reach statistical significance. In the xenograft model, mean tumor size in the room air, 2% sevoflurane, and 4% sevoflurane groups was 7.1 ± 1.9, 2.7 ± 2.0, and 2.1 ± 0.9 cm³, respectively (p = 0.041 for room air vs. 2% sevoflurane; p = 0.034 for room air vs. 4% sevoflurane). Tumor weight was likewise lower in the sevoflurane groups (room air, 7.88 ± 2.2 g; 2% sevoflurane, 2.95 ± 2.1 g; 4% sevoflurane, 2.3 ± 1.6 g; p = 0.044 for room air vs. 2% sevoflurane; p = 0.067 for room air vs. 4% sevoflurane). No statistically significant differences in tumor HIF-1α protein levels were observed among the three groups. Conclusions: In this exploratory study, sevoflurane exposure was associated with reduced HepG2 xenograft tumor growth in vivo, whereas its in vitro effects were more limited: a reduction in viable cell number was observed only at 2% sevoflurane, and an effect on cell migration could not be confirmed when analyzed across experiments. Tumor HIF-1α levels did not differ significantly between groups, suggesting that other molecular pathways may be involved. Further mechanistic and clinical studies are warranted before any conclusions can be drawn about the relevance of these findings to the perioperative management of patients with HCC. Full article

Taxanes represent a crucial class of chemotherapeutic agents clinically employed for the treatment of various malignancies, including breast cancer. Three commonly utilized taxanes—paclitaxel (Taxol), docetaxel (Taxotere), and cabazitaxel—are substantially limited by inherent drawbacks such as poor aqueous solubility, rapid clearance, and non-specific distribution. To enhance their anti-tumor efficacy against breast cancer, an mPEG-PLA-(LA)₄ nanomicelle carrier was designed and synthesized in this study for the loading of these three taxanes. These nanomicelles were designed to improve the aqueous compatibility and nanomicelle-mediated delivery of hydrophobic taxanes. In vivo animal experiments were conducted to evaluate the therapeutic effects of the three drug-loaded nanomicelles on subcutaneous human breast cancer MCF-7 xenografts in BALB/c nude mice. The results demonstrated that the injectable paclitaxel, docetaxel, and cabazitaxel micelles exhibited significant inhibitory effects on the MCF-7 xenograft tumors. These findings suggest that mPEG-PLA-(LA)₄ DS nanomicelles may serve as a structurally defined and versatile carrier platform for hydrophobic taxane delivery. Full article

Background/Objective: This study aimed to use multiple disk domain receptor 1 (DDR1), transforming growth factor β1 (TGFβ-1), tumor-associated calcium signal transduction protein 2 (TACSTD2), and polyligand proteoglycan 1 (SDC1) siRNA to treat pancreatic cancer with the goals of high specificity, significant therapeutic efficacy, and relatively low toxicity. Methods: (1) A microfluidic method was used to prepare siRNA-LNPs with different formulations. (2) Quantitative PCR (qPCR) and Western blot assays were used to detect the inhibitory effect of different-prescription siRNA-LNP formulations on mRNA and protein expression levels of related genes in PaTu 8988 pancreatic cells. (3) The anti-pancreatic cancer effect of multiple siRNAs combined with LNPs in vivo was evaluated using the BALB/c nude mouse model with subcutaneous pancreatic cancer xenografts. Results: (1) Three siRNA-LNP formulations, DMG, CE 1.5, and CE 0.75, were successfully prepared, exhibiting small particle sizes and uniform distribution. (2) qPCR and Western blot results indicated that DDR1, TGFβ-1, TACSTD2, and SDC1 siRNA-LNP significantly inhibited related genes’ mRNA and protein expression in pancreatic cancer PaTu 8988 cells. (3) Efficacy studies in animals indicated that multiple siRNA combined with LNPs in each group exhibited significant antitumor effects on pancreatic cancer tumor-bearing nude mice. The therapeutic efficacy of the combined siRNAs was superior to that of single siRNA treatments, indicating a clear combined effect, especially with three- and four-siRNA combinations. Conclusions: The prepared DDR1/TGFβ-1/TACSTD2/SDC1 siRNA-loaded LNP demonstrated a small particle size, high gene inhibition efficiency, and a significant therapeutic effect in treating pancreatic cancer. Its safety is generally acceptable, but attention should be paid to the toxicity caused by LNP excipients, especially cationic lipids. Full article

Salivary adenoid cystic carcinoma (SACC) is a malignant salivary gland neoplasm characterized by aggressive local invasion and a marked propensity for metastasis. However, the role of MEOX1 in SACC progression remains poorly defined. In this study, we examined the effects of MEOX1 overexpression on the malignant behavior of SACC cells in vitro and in vivo. Human SACC-83 and SACC-LM cells were transduced with lentiviral vectors encoding MEOX1 or an empty vector control, and cell proliferation, migration, invasion, and cell cycle distribution were assessed using CCK-8, wound healing, Transwell, and flow cytometric assays, respectively. RNA sequencing was performed to characterize transcriptional changes associated with MEOX1 overexpression. In vivo, tumor growth was evaluated in BALB/c nude mice bearing subcutaneous xenografts, and pulmonary metastatic colonization was assessed using a tail vein injection model. MEOX1 overexpression reduced the proliferation, migration, and invasion of SACC cells in vitro and increased the G2/M phase fraction. In xenograft models, MEOX1-overexpressing cells formed smaller tumors and showed lower Ki67 staining than control cells. In the experimental lung metastasis model, mice injected with MEOX1-overexpressing cells developed fewer pulmonary metastatic nodules. RNA-seq identified 588 differentially expressed genes associated with MEOX1 overexpression, with enrichment in pathways including cytokine–cytokine receptor interaction, Toll-like receptor signaling, and G protein-coupled receptor signaling. Together, these findings indicate that enforced MEOX1 expression is associated with reduced malignant phenotypes in SACC models and with transcriptomic alterations in pathways related to immune response, G protein-coupled receptor signaling, and DNA damage response. Full article

This study aimed to investigate the wound-healing mechanisms of chitosan with a defined molecular weight (MW) and degree of deacetylation (DD), and to explore its potential in hydrogel formulations, optimized for enhanced antibacterial performance. An active molecular chitosan (AMC) was prepared via enzymatic treatment to target a specific MW range with excellent biological activity. The antibacterial, anti-inflammatory, and wound-healing effects of AMC-based hydrogels were evaluated. Given AMC’s antibacterial activity against vancomycin-resistant Staphylococcus aureus (VRSA), its anti-inflammatory effects were also evaluated in full-thickness wounds in BALB/c nude mice. Anti-inflammatory effects were assessed using ELISA and immunohistochemical staining to measure levels of IL-1β, IL-4, IL-6, IL-10, and TNF-α. AMC treatment significantly reduced wound size and suppressed inflammatory cytokine production. These results suggest that hydrogels containing AMC may enhance both antibacterial and anti-inflammatory properties, potentially promoting wound healing. Full article

Since MDCK cells are inherently tumorigenic, their safety in vaccine production has long been a concern; thus, establishing a screening method for low-tumorigenic cells is of great significance for influenza vaccine development. This study successfully obtained a low-tumorigenic MDCK cell line through monoclonal screening and systematically evaluated its potential as a cellular substrate for influenza vaccines using male nude mice (BALB/c nu/nu, 4–7 weeks old) for tumorigenicity assessment. Comprehensive analysis of the biological characteristics of the screened cells—including growth curves and transcriptomic features—showed that the cell line exhibits stable growth and consistent traits. Transcriptomic comparison was performed between two defined biological states: parental MDCK cells (SQ group) and the low-tumorigenic clone MDCK-20B9 (SH group). Transcriptomic analysis revealed good dispersion among samples and an overall consistent gene expression distribution. Differential expression analysis identified a total of 2198 differentially expressed genes, including 902 upregulated and 1296 downregulated genes. GO functional enrichment analysis indicated that these genes are mainly involved in biological processes such as acute-phase response, retinol metabolism, mitotic chromosome condensation, and cell migration; are enriched in cellular components such as kinetochores and the extracellular matrix; and are associated with molecular functions including calcium ion binding and the Wnt signaling pathway. KEGG pathway analysis further revealed that the differentially expressed genes are significantly enriched in key pathways such as cancer pathways, cell cycle, and cell adhesion molecules. The expression trends of five key differentially expressed genes were validated by RT-qPCR. In summary, this study successfully screened a stable and consistent low-tumorigenic MDCK cell line, providing a theoretical basis and practical foundation for its use as a cellular substrate in influenza vaccine development. Full article

5-Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is one of the treatment modalities for oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC). However, the role of ferroptosis in ALA-PDT for OLK and OSCC remains unclear. Therefore, this study aimed to investigate whether ALA-PDT can induce ferroptosis in OLK and OSCC. We detected relative cellular dehydrogenase activity (CCK-8 assay), long-term proliferative viability, reactive oxygen species (ROS) generation, glutathione levels, and mitochondrial morphology after ALA-PDT. The expression of ferroptosis-related proteins was detected using Western blot. A tongue OSCC model was established in male BalB/c nude mice, and then ALA-PDT was performed. Immunohistochemical staining of Ki67, GPX4 and FTH1 was conducted to evaluate the effect of ALA-PDT. Subsequently, OLK and OSCC cells were pre-treated with ferrostatin-1 (Fer-1) before ALA-PDT. Relative cellular dehydrogenase activity, ROS generation, lipid peroxidation, Fe²⁺ levels, and ferroptosis-related protein expression were measured. Finally, OLK and OSCC cells were treated with a combination of ALA-PDT and erastin, and mitochondrial function was evaluated. In vitro study showed that ALA-PDT increased ROS generation and decreased GSH/GSSG ratio in OLK and OSCC cells. After ALA-PDT, mitochondrial morphology exhibited typical characteristics of ferroptosis. In vivo experiments showed that immunohistochemistry (IHC) scores of Ki67, GPX4 and FTH1 in the tissues decreased after ALA-PDT. Moreover, pre-treatment with Fer-1 could reverse ROS levels, lipid peroxidation and intracellular Fe²⁺ accumulation in OLK and OSCC cells after ALA-PDT. Additionally, Fer-1 pre-treatment reversed the changes in protein expression induced by ALA-PDT. The combination of ALA-PDT and erastin significantly reduced mitochondrial O₂•⁻ production and decreased mitochondrial membrane potential. Above all, ALA-PDT can induce ferroptosis in OLK and OSCC. The use of ferroptosis agonists may enhance the therapeutic efficacy of ALA-PDT for OLK and OSCC. Full article

Background/Objectives: Breast cancer remains a major cause of cancer-related mortality among women worldwide, highlighting the need for new therapeutic strategies. Pyrimidine derivatives have shown promise in oncology due to their ability to modulate immune responses and influence tumor growth pathways. Methods: Cytotoxicity of Xymedon was evaluated using MTT and colony formation assays on cancer MCF-7, NCI-H322M, HCT-15 cells, and primary human foreskin fibroblasts. In vivo efficacy was assessed in an orthotopic MCF-7 xenograft model in female Balb/c nude mice. Xymedon was administered orally at 410 mg/kg daily alone or in combination with intraperitoneal doxorubicin (1 mg/kg weekly). Hematological, histological, and immunohistochemical analyses were performed. Results: In vitro, Xymedon (up to 3 mM) showed no cytotoxicity against cancer cell lines or human skin fibroblasts. In vivo, Xymedon significantly increased tumor necrosis (44.1% vs. 28.5%, p < 0.01) and enhanced intratumoral infiltration of CD3+, CD8+, and CD20+ lymphocytes, with peritumoral counts increasing 2.2–5.3-fold. It mitigated Doxorubicin-induced myelosuppression by improving red blood cell counts, hemoglobin, and hematocrit levels, while platelet recovery remained limited. Combination therapy with Xymedon did not affect tumor volume or weight, but resulted in a non-significant trend toward improved survival (80% vs. 30%, p ≈ 0.11; Hazard Ratio [HR] = 0.268, 95% CI: 0.07082 to 1.012) without affecting fibrous capsule formation. Conclusions: These results suggest that Xymedon is a non-cytotoxic immunomodulator with potential as an adjuvant to enhance antitumor immunity and reduce hematologic toxicity associated with chemotherapy. Further studies are needed to elucidate the molecular pathways and confirm clinical efficacy. Full article

Background/Objectives: Targeting the PI3K/mTOR pathway is a promising strategy in cancer therapy, but its efficacy is often limited by apoptosis resistance. This study investigates the dual PI3K/mTOR inhibitor YYN-37, exploring its capacity to induce context-dependent cell death, particularly the non-apoptotic process of methuosis. Methods: We examined the effects of YYN-37 on HCT-116 and SJSA-1 cancer cell lines using cell viability assays, Western blot, and fluorescent tracers. Cell death mechanisms were probed with pathway-specific inhibitors. The role of VPS34 was assessed through kinase activity assays, siRNA-mediated knockdown, and rescue experiments with the agonist leucine. Proteomic profiling and an in vivo SJSA-1 xenograft model in BALB/c nude mice were utilized to evaluate broader mechanisms and anti-tumor efficacy. Results: YYN-37 induced caspase-3-dependent apoptosis in HCT-116 cells. In contrast, it triggered a reversible, cytoplasmic vacuolization in SJSA-1 cells, identified as methuosis. This vacuolization originated from endocytic pathways and was inhibited by EIPA and Baf-A1. YYN-37 directly inhibited VPS34 (IC₅₀ = 2.73 nM), and its knockdown replicated the vacuolization, which was conversely reversed by the VPS34 agonist leucine, confirming VPS34-dependency. Proteomics revealed lysosomal dysfunction in SJSA-1 cells and cell cycle alterations in HCT-116 cells. In vivo, YYN-37 treatment resulted in a 72.71% tumor growth inhibition, with histology confirming methuosis-like vacuolization. Conclusions: YYN-37 exerts potent, context-dependent anti-tumor effects by inducing apoptosis in HCT-116 cells and VPS34-mediated methuosis in SJSA-1 cells. This work establishes methuosis induction as a viable therapeutic strategy for apoptosis-resistant cancers and highlights VPS34 inhibition as a promising mechanism of action. Full article

Background: The immunocheckpoint TIM-3 is also expressed on acute myeloid leukemia (AML) blasts. Its prognostic significance requires clarification through subgroup analysis, while its functional roles and underlying mechanisms remain to be further investigated. Methods: Expression of TIM-3 was assessed in fresh bone marrow samples from 81 newly diagnosed patients with AML and 7 healthy donors using multi-color flow cytometry. TIM-3 overexpression was induced in Kasumi-1 and HL60 cell lines via lentiviral infection, and subsequent assays for cell proliferation, cell cycle, apoptosis, subcutaneous tumor formation, and Western blotting were performed. Sorted CD34⁺ cells from bone marrow mononuclear cells of 4 newly diagnosed AML patients were used for evaluating Ki67⁺ frequency with TIM-3 blocked or not. CD34⁺ cells from bone marrow mononuclear cells of other 4 newly diagnosed patients with AML were sorted into TIM-3⁺ and TIM-3⁻ cells and subjected to transcriptome sequencing. Results: High frequencies of CD34⁺TIM-3⁺ cells at diagnosis were related to high relapse rates in AML patients with t(8;21) (p = 0.025) but not in non-CBF-AML patients (p = 0.16). In vitro, TIM-3 upregulation in Kasumi-1 and HL60 cells enhanced cell proliferation (p = 0.002 and 0.013) and increased the S phase cell population (p = 0.006 and < 0.001), without affecting apoptosis (all p > 0.05). In vivo, TIM-3 upregulation promoted subcutaneous tumor formation in BALB/c nude mice, particularly in t(8;21) AML cells (p = 0.0068 and 0.045). In addition, blocking TIM-3 tended to decrease Ki-67⁺ frequency in CD34⁺ cells of AML patients (p = 0.058). KEGG enrichment analysis of transcriptome data revealed significant enrichment of cell cycle, with key genes including CDK1, CCNA2, CDCA5, AURKB, SGO1, TTK, TICRR, and NDC80 showing significantly higher expression in CD34⁺TIM-3⁺ cells compared to CD34⁺TIM-3⁻ cells. Notably, CDK1 and CCNA2, critical regulators of the cell cycle, were upregulated in TIM-3-overexpressing Kasumi-1 and HL60 cells. Conclusions: High TIM-3 expression in AML blasts at diagnosis is associated with relapse in the t(8;21) subtype. TIM-3 promotes AML blast proliferation by upregulating CDK1 and CCNA2, facilitating cell cycle entry. Full article

Cervical cancer ranks as a primary contributor to cancer-related deaths in women globally and is the fourth most prevalent malignant neoplasm. Cepharanthine, a naturally occurring biscoclaurine alkaloid extracted from Stephania cepharantha, has demonstrated anticancer and antimetastatic efficacy across multiple cancer types. However, its mechanism of action in cervical cancer remains unexplored. Our results demonstrated that cepharanthine effectively suppressed the proliferation and motility of the CaSki, HeLa, and C33A cell lines. Furthermore, cepharanthine triggered apoptosis through Bcl-2 suppression and increased cleaved-PARP-1, Bax, and cleaved-caspase-3 expression and AMPK/p53 phosphorylation, while inducing G0/G1 phase arrest in CaSki cells and sub-G1 phase arrest in HeLa and C33A cells. Additionally, cepharanthine reduced the mitochondrial membrane potential (∆ψm), compromised mitochondrial functionality, and increased reactive oxygen species (ROS) accumulation, promoting oxidative stress via the modulation of the Nrf2/Keap1 pathway in CaSki, HeLa, and C33A cells, which exhibit an anti-cervical cancer effect. Similarly, cepharanthine markedly reduced tumor progression in C33A BALB/c nude mice, which aligns with the in vitro observations. Collectively, these findings indicate that cepharanthine has potential therapeutic applications in the treatment of cervical cancer and warrants future clinical investigation. Full article

Objectives: This exploratory preclinical study aimed to compare the correlations of apparent diffusion coefficient (ADC) and native T1 mapping histogram features with tumor cell proliferation, microvessel density (MVD), and extracellular matrix composition in neuroblastoma xenografts. Methods: Neuroblastoma xenografts (n = 42) were established by subcutaneously injecting three MYCN-amplified/non-amplified human neuroblastoma cell lines (IMR-32, SK-N-BE(2), and SH-SY5Y; n = 14 per group) into female immunodeficient BALB/c-nude mice. Once tumors reached a diameter within the range of 12–15 mm, native T1 mapping and diffusion-weighted imaging were performed using a 3.0T clinical MRI scanner. Tumor cell proliferation and MVD were assessed via immunohistochemical Ki-67 staining and CD31 staining, respectively. Collagen fibers were visualized using Masson staining to calculate the collagen volume fraction (CVF). Pearson correlation coefficients with false discovery rate (FDR) correction were used to evaluate their associations. Results: Significant negative correlations were observed between Ki-67 expression and multiple ADC values after FDR correction, including ADC_10Percentile (r = −0.397, adjusted p = 0.032), ADC_90Percentile (r = −0.394, adjusted p = 0.032), ADC_maximum (r = −0.362, adjusted p = 0.048), ADC_mean (r = −0.421, adjusted p = 0.032), ADC_median (r = −0.422, adjusted p = 0.032), ADC_minimum (r = −0.390, adjusted p = 0.032), and ADC_{rootmeansquared} (r = −0.419, adjusted p = 0.032). In contrast, multiple T1 mapping features showed significant positive correlations with CVF (adjusted p < 0.05). Conclusions: ADC and T1 mapping provide complementary insights into tumor proliferation and extracellular matrix composition in neuroblastoma. These preclinical findings support further research to validate their potential clinical utility. Full article

The potential of N-terminal mitochondrial-targeting sequences (MTSs) as potent antimicrobial peptides (AMPs) has been previously reported. Building on this, 3923 mitochondrial proteins were identified from various marine organisms, among which 470 MTSs were predicted using MitoFates. Of these, 25 MTSs were synthesized and assessed for antimicrobial activity. All MTSs exhibited antifungal activity against Candida albicans, while 22 and 20 MTSs demonstrated activity against Escherichia coli and Staphylococcus aureus, respectively. Notably, the MTS of methylcrotonyl-CoA carboxylase subunit 1 (MCCC1-MTS) derived from swimming crab (Portunus trituberculatus) and the MTS of dihydrolipoamide branched-chain transacylase E2 (DBT-MTS) derived from herring (Oncorhynchus keta) showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi. In addition, MCCC1-MTS markedly reduced the viability of multiple cancer cell lines with minimal cytotoxicity toward HaCaT cells and effectively suppressed the growth of A549-xenografted tumors in BALB/c nude mice without inducing weight loss. These findings demonstrate that MTSs derived from marine organisms function as potent AMPs with selective cytotoxicity toward cancer cells, further supporting previous evidence that protozoan MTSs represent novel AMP candidates. Full article

Background: Gefitinib is a first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) targeting EGFR-mutated non-small cell lung cancer (NSCLC) and is a current first-line treatment for NSCLC. However, acquired resistance leads to the failure of treatment and remains a challenge. Therefore, identifying novel therapeutic approaches to combat EGFR-TKI resistance is crucial. Methods: The Cancer Genome Atlas (TCGA) database analysis and gefitinib-resistant cell lines were used to analyze VDR expression in NSCLC. Cell proliferation and apoptosis were assessed via MTT assay, colony formation assay, and flow cytometry. Immunofluorescence, qPCR, and Western blotting were used to measure mRNA and protein expression levels of VDR and other related molecules. Xenograft tumors in BALB/c nude mice were employed to investigate the effects of VDR and 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) on gefitinib-resistant tumors in vivo. Results: We found that VDR was significantly upregulated in EGFR-TKI-resistant NSCLC cells. Patients with high VDR expression exhibited poor prognosis. VDR knockdown significantly inhibited cell proliferation, tumor growth, and reduced gefitinib resistance, whereas VDR overexpression enhanced resistance. VDR knockdown downregulated EGFR and FASN expression. Silencing either EGFR or FASN confirmed the existence of a positive feedback regulatory loop involving VDR, EGFR, and FASN. Treatment with 1,25(OH)₂D₃ increased VDR levels but decreased EGFR and FASN expression. VDR knockdown significantly enhanced the inhibitory effect of 1,25(OH)₂D₃ on gefitinib resistance. The combination of VDR knockdown and 1,25(OH)₂D₃ treatment was more effective than either treatment alone in suppressing EGFR and FASN expression. Conclusions: VDR promotes NSCLC resistance to EGFR-TKIs by regulating EGFR and FASN expression through a positive feedback loop. Knocking down VDR effectively enhances the ability of 1,25(OH)₂D₃ to overcome gefitinib resistance, mediated by the synergistic downregulation of EGFR and FASN expression. Targeting VDR represents a potential strategy to enhance the efficacy of 1,25(OH)₂D₃ in overcoming EGFR-TKI resistance. Full article