Curcumin Enhances the Sensitivity of 5-fluorouracil-resistant Colon Cancer Cells to 5-fluorouracil Through a Ferroptosis-related Mechanism: A Network Pharmacology and In Vitro Study

Abstract

Colorectal cancer (CRC) is a common and deadly malignancy. Although 5-fluorouracil (5-FU) continues to be a standard therapeutic agent for CRC, the development of resistance substantially compromises its clinical effectiveness. Curcumin, a natural polyphenol, has shown potential in reversing chemoresistance, possibly through ferroptosis-related mechanisms. The present work explored the potential of curcumin to restore responsiveness to 5-FU in resistant CRC cells and examined the possible involvement of ferroptosis through a combined bioinformatics and experimental strategy. Curcumin-related targets were predicted using SwissTargetPrediction, Super-PRED, and Herbal Ingredients’ Targets (HIT). Protein–protein interaction (PPI) networks, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, as well as ferroptosis-related target screening, were performed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Metascape, and FerrDb. Genes associated with CRC and 5-FU resistance were retrieved from GeneCards and Genomics of Drug Sensitivity in Cancer (GDSC) databases, and intersecting candidates were subsequently mapped using Cytoscape software. In vitro experiments were conducted in HCT8/5-FU cells. Cellular proliferation and viability were evaluated with the Cell Counting Kit-8 (CCK-8) method. Boron-dipyrromethene (BODIPY) 581/591 C11 staining was performed to evaluate lipid peroxidation, whereas intracellular ferrous iron (Fe 2+ ) accumulation was determined by flow cytometric analysis. Expression levels of epidermal growth factor receptor (EGFR) and glutathione peroxidase 4 (GPX4) were evaluated by Western blot and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Ferrostatin-1 (Fer-1) was used to investigate the involvement of ferroptosis. Network pharmacology analysis showed that curcumin targets genes and pathways associated with chemoresistance and ferroptosis, with EGFR identified as a key target. In vitro experiments demonstrated that curcumin reduced the proliferative capacity of HCT8/5-FU cells and enhanced the inhibitory effect of 5-FU. Curcumin increased lipid reactive oxygen species (ROS) and intracellular Fe 2+ levels, accompanied by reduced mRNA and protein activity of GPX4 and EGFR. Fer-1 partially restored cell viability and reversed the downregulation of ferroptosis-related markers, while intracellular Fe 2+ remained elevated. Curcumin restored responsiveness to 5-FU in resistant CRC cells and may be associated with ferroptosis-related mechanisms involving lipid peroxidation, iron metabolism, and EGFR regulation. These findings provide credence to the idea that curcumin could be a useful supplementary tactic for overcoming chemoresistance in CRC.