Integrated bioinformatics and experimental validation identifies the antimalarial drug mefloquine as a ferroptosis inducer in colorectal cancer via mitochondrial damage and oxidative stress-mediated lipid peroxidation

Colorectal cancer (CRC) ranks as the third most frequently diagnosed malignancy and the second leading cause of cancer-related mortality worldwide. Despite advances in multimodal therapy, effective diagnostic biomarkers and therapeutic agents remain limited. This study aimed to identify CRC-associated marker genes and screen candidate repurposable drugs. Four GEO microarray datasets and TCGA RNA-sequencing data (382 CRC samples) were integrated using weighted gene co-expression network analysis (WGCNA), support vector machine recursive feature elimination (SVM-RFE), Cox proportional hazards regression, and Lasso to identify marker genes associated with occurrence, development, and prognosis. Connectivity Map (CMap) analysis nominated candidate drugs, which were functionally validated in vitro (CCK-8, flow cytometry, Transwell, wound healing) and in vivo (HT29 xenograft model). Underlying mechanisms were delineated via transcriptomic profiling, Seahorse metabolic flux analysis, Western blotting, immunofluorescence, and transmission electron microscopy. Twenty CRC marker genes were identified (8 occurrence-related, 11 development-related, 6 prognosis-related). CMap analysis nominated ten repurposable drugs, among which mefloquine exhibited the most potent and broad-spectrum antiproliferative activity (IC50 < 10 μM across all seven tested CRC cell lines). In vivo , oral mefloquine at 50 mg/kg reduced xenograft tumor weight by 54.5% ( p < 0.01) without appreciable toxicity. Mechanistically, mefloquine suppressed mitochondrial oxidative phosphorylation (basal OCR reduced by 67%, ATP by 72%), triggered mitochondrial ROS accumulation (22.9% to 47.4%), depleted glutathione, elevated lipid peroxidation (4-HNE and MDA ~ 2-fold), and increased intracellular iron (~3-fold), culminating in ferroptotic cell death. These findings identify mefloquine as a potent ferroptosis inducer in CRC via mitochondrial impairment and oxidative stress-mediated lipid peroxidation, supporting its repurposing as a novel therapeutic agent for colorectal cancer.