Unveiling Gloriosine as a Dual-Acting Regulator of Glutamine Metabolism and Ferroptosis in Triple-Negative Breast Cancer: Insights from Network Pharmacology and Experimental Validation

Abstract Background TNBC lacks clearly defined molecular targets, so chemotherapy remains the standard approach despite resistance, toxicity, and high relapse rates. New, less toxic therapeutic options are urgently needed. This study evaluated the anticancer potential of gloriosine, a plant-derived alkaloid known to inhibit tumor cell growth with minimal effect on normal breast cells. Methods Putative targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, then intersected with gene sets linked to TNBC and glutamine metabolism. The resulting network was characterized through protein-protein interaction mapping and Gene Ontology/KEGG enrichment analysis. Molecular docking assessed binding affinity to top targets, and predictions were validated experimentally using cell viability, colony formation, and wound-healing assays. Oxidative stress and ferroptosis were assessed via ROS (DCFDA), glutathione, and lipid peroxidation (MDA) assays, along with Western blotting and FerroOrange staining. Results Network analysis identified 100 predicted targets, 60 overlapping with TNBC/glutamine-metabolism genes; SRC, EGFR, mTOR, and HSP90AA1 emerged as hub proteins. Enrichment analysis linked this network to cancer progression, metabolic reprogramming, and resistance pathways, including central carbon metabolism and ErbB/EGFR-inhibitor resistance signaling. Docking confirmed strong gloriosine-target binding. Experimentally, gloriosine reduced cell proliferation and migration in a dose-dependent manner. Mechanistically, it suppressed glutaminolysis and induced ferroptosis, marked by increased ROS, glutathione depletion, elevated lipid peroxidation, GPX4 suppression, and intracellular iron accumulation. Conclusions Gloriosine suppresses TNBC growth via multi-target modulation and ferroptosis induction, supporting its potential as a novel anticancer candidate. Keywords: Triple-negative breast cancer (TNBC), gloriosine, network pharmacology, ferroptosis.