Search Results (3,633)

Background: Photodynamic therapy (PDT) is a cancer treatment that combines a photosensitizer (PS), light, and oxygen to generate reactive oxygen species (ROS), leading to tumor cell death. PDT efficacy depends largely on PS accumulation within tumors, prompting the development of third-generation PSs and nanotechnology-based delivery systems. Among these, BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are promising PSs due to their favorable photophysical properties, while liposomes improve drug delivery, cellular uptake, and sustained release profiles. This study describes the synthesis of two novel BODIPY derivatives differing in the position of a methyl ester group on the meso-phenyl ring, their incorporation into liposomes, and evaluation of PDT efficacy. Methods: Cellular uptake of BODIPY-loaded liposomes, intracellular ROS generation, apoptosis, necrosis, and lipid peroxidation were assessed by flow cytometry in colorectal and ovarian cancer cell lines. The antitumor activity of the liposomal formulations was further evaluated in both 2D and 3D models using MTT and clonogenic assays. The involvement of ferroptosis and necroptosis in PDT-induced cell death was also investigated. Results: Liposomal formulations significantly enhanced cellular uptake compared with free compounds. Following light activation, both formulations induced potent antitumor effects through multiple cell death mechanisms, including canonical and non-canonical pathways, and maintained strong efficacy in 3D tumor spheroids. Conclusions: Liposome-encapsulated BODIPYs represent promising PDT agents by improving cellular uptake and eliciting robust antitumor activity through complementary cell death mechanisms. Furthermore, the methyl ester substituent on the meso-phenyl ring provides a versatile platform for future conjugation with targeting ligands, supporting the development of third-generation, tumor-targeted photosensitizers and warranting further preclinical investigation. Full article

Cellular senescence plays a complex and crucial dual role in ovarian cancer treatment: it serves as both an important tumor suppression mechanism and a potential driver of drug resistance and recurrence. This review systematically examines the intricate regulatory network of cellular senescence, encompassing multiple pathways including telomere dysfunction, DNA damage response, epigenetic remodeling, hormone signaling, and metabolic reprogramming. Studies have demonstrated that core therapeutic modalities, such as platinum-based chemotherapy, PARP inhibitors, and CDK4/6 inhibitors, can exert anti-tumor effects by inducing cellular senescence; however, their efficacy is significantly influenced by the senescence-associated secretory phenotype (SASP). Specific SASP components can activate immune surveillance, whereas others promote tumor progression, acquisition of stem cell-like characteristics, and therapeutic resistance. It is this “anti-tumor versus pro-tumor” paradox that renders senescent cells a critical variable determining treatment outcomes. In light of this, emerging strategies aim to precisely modulate the senescence process, including the use of epigenetic drugs to induce senescence, targeting metabolic vulnerabilities to eliminate senescent cells, and intervening in deleterious SASP to improve the tumor microenvironment. Integrating such strategies with conventional therapies holds promise for overcoming the resistance bottleneck in ovarian cancer and opening new avenues for improving patient prognosis. Full article

Zoledronic compounds are widely used as antiresorptive and antitumor agents to prevent bone loss and treat multiple myeloma; however, their pharmacokinetic limitations motivate the search for alternative delivery systems capable of improving their controlled release and bioavailability. In this work, we investigate the stability, structural behavior, and adsorption properties of zoledronic acid (ZOL) and its Ca²⁺ and Zn²⁺ salts confined within the interlayer space of the smectite clay mineral montmorillonite by combining empirical force field (FF), density functional theory (DFT), and molecular dynamics (MD) simulations. The main objective of this study is to evaluate the suitability of montmorillonite as a potential drug delivery system (DDS). Specifically, crystal polymorph structures of zoledronic acid [1-(2-hydroxy-2-phosphonate-2-phosphonoethyl)-1H-imidazol-3-ium)] (ZOL) and its Ca²⁺ and Zn²⁺ salts were analyzed. Our calculated crystal structures obtained by both methods (FF and DFT) agree well with the known experimental data. Furthermore, the intercalation of ZOL into the confined interlayer space of montmorillonite is energetically favorable. Several interlayer cations (Na⁺, Ca²⁺, and Zn²⁺) were also evaluated. MD simulations showed that ZOL adopts stable confined configurations within the interlayer space of montmorillonite, exhibiting small torsions of the imidazole group. Additionally, the desorption of ZOL in a modelized acidic medium is energetically favorable. Our calculations predict that this clay mineral holds strong potential for the controlled delivery of zoledronic compounds. Full article

Triple-negative breast cancer (TNBC) remains a highly aggressive malignancy with limited therapeutic options and frequent resistance to platinum-based chemotherapy. Rosmarinic acid (RA), a naturally occurring polyphenol, has attracted considerable interest as a potential chemosensitising agent. This study investigated the anticancer activity and the underlying mechanisms of RA combined with cisplatin (CDDP) in 4T1 breast cancer cells while assessing the cytotoxic responses of non-cancerous HaCaT keratinocytes as a preliminary indicator of differential treatment sensitivity. Cytotoxicity was assessed using the MTT assay, followed by calculation of the Combination Index (CI), Drug Reduction Index (DRI), and Selectivity Index (SI). The generation of intracellular reactive oxygen species (ROS) was evaluated by DCFH-DA fluorescence imaging, and the functional contribution of oxidative stress was examined using N-acetyl-L-cysteine (NAC) rescue experiments. Apoptosis was analysed by Annexin V/PI flow cytometry, NucBlue nuclear staining, and Calcein-AM/propidium iodide (PI) Live/Dead fluorescence imaging. Three-dimensional (3D) tumour spheroids were used to assess treatment-induced alterations in spheroid morphology, morphometric parameters, viability based on adenosine triphosphate (ATP), and Live/Dead staining. The expression of genes related to apoptosis was determined by RT-qPCR, and potential molecular mechanisms were explored using the construction of protein–protein interaction (PPI) networks together with Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses. The combination of RA + CDDP exhibited strong synergistic cytotoxicity in 4T1 cells while demonstrating comparatively lower toxicity toward HaCaT keratinocytes. Combination treatment markedly increased intracellular ROS generation, whereas NAC significantly reduced ROS accumulation and partially restored cell viability, indicating that oxidative stress is a major but not exclusive mediator of cytotoxicity. Combined treatment significantly enhanced apoptotic cell death, increased chromatin condensation and membrane damage, upregulated the expression of Bax, Casp9, Cycs, and Trp53, and downregulated Bcl2, consistent with transcriptional regulation of intrinsic apoptotic signalling. In 3D tumour spheroids, the combination markedly reduced spheroid size, disrupted structural integrity, decreased ATP-based viability, and substantially increased tumour cell death compared to monotherapy. Bioinformatic analyses identified central genes related to apoptosis and cell survival and predicted significant enrichment of PI3K/Akt, p53, MAPK, and apoptosis signalling pathways. RA significantly potentiates the antitumor efficacy of CDDP through synergistic induction of ROS-associated apoptotic signalling while showing a more favourable cytotoxic response in 4T1 breast cancer cells than in non-cancerous HaCaT keratinocytes. The integrated findings from two-dimensional (2D) and 3D models, NAC rescue experiments, molecular analyses, and bioinformatics collectively support the potential of RA as a promising chemosensitising adjuvant for CDDP-based breast cancer therapy and warrant further validation in preclinical in vivo models. Full article

Colorectal cancer (CRC) remains one of the leading causes of cancer-related morbidity and mortality worldwide despite substantial advances in surgery, chemotherapy, targeted therapies, and immunotherapy. The limited efficacy of current treatment strategies in advanced disease and the emergence of therapeutic resistance highlight the urgent need for novel adjunctive therapeutic approaches. Increasing evidence indicates that chronic stress and sustained activation of β-adrenergic signaling promote colorectal tumor initiation, progression, angiogenesis, metastatic dissemination, and immune evasion, thereby identifying this pathway as a potential therapeutic target. Drug repurposing has emerged as an attractive strategy for accelerating the development of new anticancer therapies by identifying novel applications for clinically approved drugs with well-established safety profiles. Among these, β-blockers have gained considerable attention because of their ability to inhibit β-adrenergic signaling and modulate multiple oncogenic pathways implicated in CRC progression. Although accumulating preclinical and observational clinical evidence suggests that β-blockers may possess anticancer potential, the underlying molecular mechanisms and their translational relevance have not yet been comprehensively integrated. This review provides a critical overview of the current evidence regarding the therapeutic potential of β-blockers in CRC by integrating findings from preclinical and clinical studies. Particular emphasis is placed on the regulation of key signaling pathways, including cAMP/PKA/CREB, PI3K/AKT/mTOR, and RAS/RAF/MEK/ERK, as well as on the effects of β-blockers on tumor cell proliferation, apoptosis, angiogenesis, epithelial–mesenchymal transition, metastasis, and modulation of the tumor microenvironment and antitumor immune responses. However, significant barriers limit the translation of these findings into routine clinical practice, including the limited representativeness of preclinical models, potential hemodynamic adverse effects, and the inherent limitations of observational studies. Importantly, owing to the lack of prospective randomized clinical trials, the current evidence remains insufficient to establish the clinical efficacy of β-blockers in CRC. Although β-blockers possess several characteristics that make them attractive candidates for drug repurposing, including a well-established safety profile, widespread availability, and low cost, further mechanistic studies, prospective randomized clinical trials, and biomarker-based patient stratification are essential to determine their clinical efficacy and define their role in personalized CRC therapy. Full article

Oxidative stress in the tumor microenvironment, which is its own intrinsic property, is frequently utilized to deal with the drug-targeting issue in the nanoparticle drug delivery system. For this purpose, reactive oxygen species (ROS)-sensitive nanoparticles encapsulating doxorubicin (DOX) and chlorin e6 (Ce6) were synthesized for treatment of MDA-MB-231 breast cancer cells. Hyaluronic acid (HA) with a reductive end was conjugated with methoxy poly(ethylene glycol) (PEG) using thioketal diamine (ThdNH₂) linkage (HA-b-PEG copolymer). Then, Ce6 were conjugated to the carboxylic acid group of HA via ThdNH₂ (HA(Ce6)-b-PEG copolymer). DOX was physically incorporated to make DOX-incorporated HA(Ce6)-b-PEG copolymer nanoparticles (DOX-NP). HA(Ce6)-b-PEG copolymer nanoparticles (empty NP) and DOX-NP have a tiny particle size, less than 200 nm, with spherical morphology. They were responsively disintegrated according to the hydrogen peroxide (H₂O₂) concentration, then the release rate of Ce6 or DOX was accelerated, indicating that empty NP and DOX-NP have ROS sensitivity. DOX-resistant MDA-MB-231 cells were prepared by continuous treatment of DOX for three months. DOX-NP were efficiently internalized into the cells while intra-cellular delivery of DOX itself was inhibited. DOX-NP has higher anticancer activity against DOX-resistant MDA-MB-231 cells than that of DOX itself since cells were resistant to DOX itself. Under light irradiation, DOX-NP significantly decreased the viability of DOX-resistant MDA-MB-231 cells while DOX itself did not properly affect cell viability. Empty NP also efficiently inhibited cell viability rather than that of Ce6 itself while both of them did not affect the cell viability in the absence of light irradiation. Furthermore, empty NP showed higher ROS generation than that of Ce6 itself. DOX-NP more efficiently induced apoptosis/necrosis than DOX itself. In DOX-resistant MDA-MB-231 cell-bearing mice, DOX-NP was efficiently delivered to tumor tissue. DOX-NP greatly inhibited the growth of tumors under light irradiation, more than that of DOX itself or empty NP. In conclusion, DOX-NP showed promising antitumor activity against DOX-resistant MDA-MB-231 cells. Full article

Background/Objectives: Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that regulates multiple oncogenic and tumor-suppressive pathways and has emerged as a promising target for anticancer drug discovery. However, most reported USP7 inhibitors belong to a limited number of structural classes, and no USP7-targeted therapy has yet reached clinical application. This study aimed to identify structurally diverse candidate USP7-inhibitory scaffolds with antiproliferative activity using an integrated computational and experimental screening strategy. Methods: A drug discovery workflow combining pharmacophore modelling, structure-based virtual screening, molecular docking, and in silico pharmacokinetic assessment was employed to identify candidate USP7 inhibitors. Selected compounds were evaluated for inhibition of recombinant USP7 and for antiproliferative activity in a panel of human cancer cell lines. Molecular docking analyses were performed to investigate predicted binding modes. Results: The primary screening campaign identified four active compounds (>50% inhibition at 10 µM) and twenty-one weakly active compounds (10–49% inhibition at 10 µM), including structurally distinct approved drugs and compounds from an in-house chemical library. The four active compounds were subsequently validated by dose–response assays against recombinant USP7 and exhibited micromolar inhibitory activity. These candidate USP7-inhibitory scaffolds also exhibited antiproliferative activity across cancer cell lines with different molecular backgrounds. Docking studies predicted binding within a pharmacologically relevant region of the USP7 catalytic cleft and revealed putative interactions with key residues involved in ligand recognition. Among the compounds evaluated, rafoxanide demonstrated the most favorable combination of predicted USP7 binding and antiproliferative activity. Conclusions: This integrated virtual screening and experimental validation approach enabled the identification of candidate USP7-inhibitory scaffolds with preliminary antiproliferative activity. The identified hits include approved drugs with previously unreported USP7 inhibitory activity, as well as underexplored scaffolds that expand the chemical space of candidate USP7-targeting molecules. These candidate scaffolds warrant further medicinal chemistry optimization, orthogonal validation, and mechanistic characterization to establish their potential as USP7-targeted anticancer agents. Full article

UDP-glucuronosyltransferases (UGTs) are a key family of phase II metabolic enzymes in humans that play a central role in maintenance of metabolic homeostasis and drug disposition by catalyzing glucuronidation of endogenous and exogenous substances. UGT-mediated metabolism of antitumor drugs plays a dual role in tumor drug resistance, emerging as a hotspot in cancer therapy. This review outlines the structural characteristics, classification system, tissue distribution, and multilevel regulation of UGTs, with a focus on their bidirectional roles in tumor drug resistance, i.e., promotion of resistance through metabolic clearance and inhibition of resistance through metabolic activation. This review also discusses strategies of multidimensional tumor resistance intervention based on UGTs, and we also discussed the challenges in the clinical translation of current UGT-targeting strategies. To date, most data on UGTs were derived from in vitro and preclinical models; clinical validation remains limited, and the dual roles of UGTs are highly context-dependent. This review article provides a perspective for comprehensive understanding of UGT-mediated tumor resistance and offers theoretical foundations and practical directions for development of novel antitumor strategies. Full article

Objective: Vasculogenic mimicry (VM), characterized by the de novo formation of microvascular-like channels derived from aggressive tumor cells without involving traditional endothelial cells, is a pivotal pathological hallmark driving extreme invasiveness and dismal prognosis in hepatocellular carcinoma (HCC). This study aimed to establish a VM-based molecular subtyping system and systematically characterize its associated biological features, thereby providing a potential framework for individualized prognostic assessment and treatment decision-making in HCC. Methods: We integrated curated VM-associated gene sets with single-cell RNA sequencing data to identify malignant epithelial cell-enriched VM-associated candidate genes. Subsequently, univariate Cox regression, LASSO-Cox regression, and multivariate Cox regression were sequentially performed to identify six prognostic VM-related genes: HSPA9, TGFA, MAD2L1, PROM1, AGXT, and GCGR. HCC patients were stratified into VM, Mixed-VM, and Non-VM subtypes according to VM scores. Kaplan–Meier survival analysis, time-dependent ROC analysis, and Cox regression were used to assess prognostic performance. The biological features of the classification system were evaluated using bulk transcriptomic cohorts, spatial transcriptomics, Cytometry by Time-of-Flight (CyTOF), metabolomics, lipidomics, somatic mutation and copy number alteration analyses, and treatment-related HCC cohorts. Results: The VM score-based classification stratified HCC patients into three molecular subtypes with distinct prognostic and biological characteristics. Patients classified as the VM subtype had significantly poorer overall survival than those classified as Mixed-VM or Non-VM subtypes, and this prognostic pattern was validated across independent cohorts. Multi-omics analyses showed that the VM subtype was associated with YAP-TAZ-TEAD-related transcriptional programs, stemness/proliferation-related features, immunoregulatory and exhaustion-like tumor microenvironmental characteristics, and distinct metabolic and lipidomic alterations involving modified nucleosides, keto acid-related metabolites, cholesteryl esters, and sphingolipid-related species. Spatial transcriptomics revealed focal enrichment of VM-score-high regions and their association with YAP-TAZ-TEAD and immune checkpoint-related signatures. In orthotopic HCC mouse models, YAP1 overexpression increased PAS⁺/CD34⁻ VM-like structures, whereas verteporfin treatment reduced these structures. In two treatment-related cohorts, the Non-VM subtype showed higher response rates to sorafenib and transarterial chemoembolization (TACE) than the VM subtype. Connectivity Map (CMap)-based computational drug prioritization and molecular docking analysis prioritized ivermectin as a candidate compound; however, its antitumor activity requires further experimental validation. Conclusions: This study establishes a VM score-based molecular classification framework for HCC and identifies VM-subtype-associated prognostic, spatial, immune, metabolic, genomic, and therapeutic features. These findings provide a candidate framework for molecular risk stratification and subtype-guided therapeutic exploration in HCC. Full article

Glioblastoma (GB), the most prevalent and aggressive brain tumor, remains one of the most lethal challenges in modern oncology. Standard therapy remains largely ineffective, mainly due to limited therapeutic penetration across the blood–brain barrier (BBB) and adaptive tumor resistance. Consequently, there is an urgent need for innovative strategies to enhance therapeutic precision and efficacy. To address these limitations, we engineered a targeted peptide-based co-delivery system using the WRAP5 cell-penetrating peptide functionalized with a transferrin receptor (TfR)-targeting T7 peptide (sequence: HAIYPRH), enabling simultaneous delivery of temozolomide (TMZ) and a p53-encoding plasmid DNA. The resulting peptide-based TMZ/p53 nanocomplexes exhibited favorable physicochemical properties, enhanced TfR-mediated cellular uptake, and targeted antitumor activity mediated through p53-induced apoptosis in two-dimensional (2D) U87 MG cell cultures. To better reproduce the structural and cellular complexity of the tumor microenvironment, a three-dimensional U87 MG spheroid model was established and optimized using a Design of Experiments (DoE) approach to improve reproducibility and physiological relevance. The developed WRAP5-based nanocomplexes induced a significant dose-dependent inhibition of growth and morphological alterations in the U87 MG spheroid model, accompanied by deep penetration and cell death throughout the spheroid. Collectively, these findings highlight the potential of this targeted and tailored nanosystem to enhance cellular transfection, enable drug/gene co-delivery, restore p53 function, and promote apoptosis, representing a promising therapeutic strategy for GB treatment. Full article

This study combines ligand- and structure-based in silico strategies to predict the inhibitory activity of natural flavonoids on the Polo-Like Kinase-1 (PLK-1) enzyme as candidate anticancer agents. This enzyme participates in mitosis and is overexpressed in cancer cells. Furthermore, it has been shown to have important implications for tumor metastasis, and its inhibitors are attractive starting points for drug development. First, classification models are developed using linear discriminant analysis and a multilayer perceptron neural network. Models with accuracy greater than 80%, validated using standard statistical performance metrics and applicability domain, are used for virtual screening identifying four compounds as potential antitumor drugs. Subsequently, the identified compounds are evaluated using a molecular docking methodology to verify their binding mode and interactions with the catalytic domain of PLK-1. Finally, the integration of molecular dynamics simulations, at 300 ns, with Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) thermodynamic calculations demonstrates that the hydroxylation pattern of ring B in the flavonol scaffold is the fundamental chemical-structural determinant of electrostatic interactions and the architecture of water-mediated networks. Among the evaluated flavonoids, myricetin showed the most favorable overall computational profile, including the highest virtual-screening score and the most favorable mean MM/GBSA estimate, supporting its prioritization for experimental evaluation as a potential PLK-1 inhibitor. The integration of these approaches offers a robust methodological framework for proposing candidates with a higher probability of success, in subsequent stages of experimental validation, reducing time and costs in the early stages of drug development. Full article

Estrogen receptor (ER)-positive breast cancer (BrCa) accounts for two-thirds of all BrCa cases worldwide. Therefore, ER-targeted endocrine therapy is the standard treatment for this disease. There has been a recent trend towards developing combination therapies using molecularly targeted drugs to improve outcomes. This study aimed to identify therapeutic targets demonstrating efficacy when combined with fulvestrant (a selective ER downregulator/degrader). We generated microRNA (miRNA) signatures from fulvestrant-treated MCF-7 cells by RNA sequencing. From the signature, we evaluated miR-374b-5p because its expression was elevated by fulvestrant treatment in MCF-7 cells. Also, in expression analysis by subtype of BrCa patients, miR-374b-5p expression was suppressed only in luminal BrCa. Ectopic expression assays revealed that miR-374b-5p attenuated the malignant phenotypes of MCF-7 cells. We searched for genes regulated by miR-374b-5p and discovered that 11 (NEK2, NUF2, HMMR, DEPDC1B, FOXM1, ELOVL6, KIF20A, NCAPH, CENPK, FAM83D, and KIAA0101) are closely involved in BrCa molecular pathogenesis. Among these target genes, we focused on forkhead box M1 (FOXM1), a transcription factor regulating cell cycle progression and division. Notably, combination therapy with fulvestrant and a FOXM1 inhibitor significantly suppressed MCF-7 cell proliferation. From the miRNA signature established in this study, we identified antitumor miR-374b-5p and its target genes and used these findings to explore candidate drugs with potential efficacy when combined with fulvestrant. Full article

ZnO nanoparticles (ZnO NPs) have been widely investigated in the biomedical field, particularly their anti-tumor efficacy. The potential of ZnO hierarchical structures (ZnO HSs) in tumor cell eradication remains largely unexplored in prostate cancer (PCa) and thyroid cancer (TC). In this study, we successfully synthesized and characterized ZnO NPs and ZnO HSs using green tea extract (Camellia sinensis) as a reducing agent and conjugation of FIT-C tracking for both ZnO NPs and ZnO HSs. UV-vis spectrophotometry, Dynamic Light Scattering (DLS), FTIR, XDR, SEM and TEM revealed significant differences in morphology between ZnO NPs and ZnO HSs. Our in vitro experiments demonstrated that SNPs were more effective on aggressive PCa and TC cell lines compared to ZnO NPs. Notably, ZnO HSs exhibited enhanced cytotoxicity in 3D tumor cell spheroid models. Mechanistically, ZnO HSs induced apoptosis through cholesterol-mediated reactive oxygen species (ROS) generation. Our in vivo study revealed no histopathological changes in major organs (liver, kidneys, spleen and lungs), emphasizing the safe administration of both ZnO NPs and ZnO HSs. Our study synthesized FITC-conjugated non-spherical ZnO nanoparticles, providing evidence for a novel treatment strategy for hormone-related cancers and prospective fluorescent-guided nanomedicine. Full article

Plant-derived exosome-like nanoparticles (PELNs) are lipid bilayer nanostructures containing endogenous lipids, proteins, nucleic acids, and phytochemicals, which have attracted increasing interest as bioinspired carriers for cancer therapy. This review evaluates how plant source, isolation, purification procedures, vesicle composition, cargo-loading strategy, and administration route shape the quality and tumor-delivery performance of PELNs. The available evidence indicates that plant source and processing are major determinants of particle size, purity, surface charge, cargo profile, and biological activity. Ultracentrifugation remains widely used but is limited by contaminant co-isolation and poor scalability, whereas density-gradient purification and size-exclusion chromatography improve purity, and ultrafiltration and tangential flow filtration offer greater potential for large-scale manufacturing. Passive incubation generally preserves vesicle integrity and is most suitable for hydrophobic small molecules, whereas electroporation, sonication, and extrusion can increase cargo loading but may cause aggregation, membrane remodeling, or loss of endogenous components. Preclinical studies suggest that PELNs can exert intrinsic antitumor effects, modulate the tumor microenvironment, improve chemotherapeutic delivery, and help overcome drug resistance. However, evidence for in vivo tumor-targeting remains less robust than evidence for cellular uptake, and direct comparisons with established nanocarriers remain scarce. Clinical translation will require standardized nomenclature and characterization, reproducible manufacturing, quantitative loading and release assays, route-specific biodistribution studies, and repeated-dose safety evaluation. These findings provide a framework for the rational development of PELNs as reproducible tumor-oriented nanocarriers. Full article

Toad poison (ChanSu), a traditional animal-derived medicine, has long been used in East Asian medical systems for the treatment of inflammatory conditions and tumor-related disorders. While bufadienolides have been extensively investigated as its major bioactive constituents, the contribution of peptide components to its antitumor effects remains largely unexplored. Here, we identified protein-derived candidate antiproliferative peptides from toad poison using an integrated proteomics and bioinformatics-guided strategy. Proteomic analysis identified 135 proteins, from which 2117 peptide sequences were generated via in silico digestion with trypsin and pepsin. Subsequent multi-step screening using PeptideRanker, AntiCP, iACP, and ACPred yielded twelve candidate peptides with predicted anticancer activity. Network pharmacology analysis suggested their potential involvement in cancer-related targets and pathways. ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) evaluation was subsequently used as a complementary assessment of drug-like and safety-related properties, further prioritizing four peptides for experimental validation, while molecular docking supported their interactions with key lung cancer-associated targets. In vitro assays demonstrated that three of the four prioritized peptides (WEAWN, NSQWG, and ACGVIGICQ) exhibited initial antiproliferative activity against human lung cancer A549 cells, though these findings should be interpreted with caution given the absence of a positive control in the MTT assay. This study provides preliminary evidence suggesting that protein-derived peptide candidates from toad poison may possess antiproliferative potential, representing an early systematic exploration of its previously unexplored peptidome as a source of candidate antiproliferative peptides warranting further pharmacological investigation. The integrated strategy presented here offers an efficient approach for the discovery of bioactive peptides from animal-derived traditional medicines. Full article