Redox proteomics combined with lipidomics and metabolomics to elucidate the mechanism of ferroptosis in colorectal cancer using human colorectal carcinoma HCT-116 cells

<p><strong>Abstract Background: Colorectal cancer (CRC) ranks as the third leading cause of cancer-related death worldwide, and the second leading cause of cancer-related deaths in New Zealand. It is frequently driven by mutations in key oncogenes and tumor suppressor genes. Current treatment modalities for CRC, offer only limited efficacy, as many chemotherapeutic agents fail to elicit consistent responses across patient populations. This clinical challenge underscores the urgent need for novel therapeutic strategies. Ferroptosis, first described in 2012, is an iron-dependent form of regulated cell death characterized by the accumulation of lipid peroxides and reactive oxygen species (ROS). Increasing evidence has demonstrated that ferroptosis selectively targets vulnerabilities in cancer cells, particularly in iron metabolism and oxidative stress pathways, making it a promising therapeutic avenue for CRC. Furthermore, modulation of ferroptosis pathways has been shown to enhance the anti-tumor efficacy of conventional chemotherapeutics. A better understanding of the initiation, propagation, and resistance mechanisms of ferroptosis in CRC is therefore critical for the development of ferroptosis-based therapeutic interventions. During ferroptosis, cysteine thiol groups serve as direct targets of ROS and act as reversible “redox switches” through oxidative modifications such as disulfide bond formation or thiol oxidation. These modifications regulate key physiological processes, including metabolic reprogramming, oxidative stress responses, and cell survival. The goal of the research is to identify novel redox-regulated proteins, lipid species, and metabolites associated with ferroptosis, generate new insights into the molecular mechanisms of ferroptosis in CRC, and ultimately contribute to the identification of potential biomarkers and therapeutic targets for CRC treatment. Methods: In this study, the human colorectal carcinoma cell line HCT-116 was used as the experimental model. Ferroptosis was induced by treatment with ferric ammonium citrate (FAC), and phenotypic changes were characterized through a combination of morphological observation under a microscope, cell counting using hemocytometer, CCK-8 metabolic activity assay, and ROS assay.</strong></p><p>To investigate the oxidative modification of cysteine thiol groups of proteins extracted from the studied cells, we employed a quantitative proteomics approach. We employed an isobaric labeling strategy using the iodoTMT6plex reagent to selectively label reversibly oxidized thiols on cysteine residues. An anti-TMT resin-based enrichment step was then applied to enrich iodoTMT-labeled peptides and reduce interference from non-target peptides during liquid chromatography tanden mass spectrometry (LC-MS/MS) analysis. Protein and peptide identification and quantification were performed using LC-MS/MS and Proteome Discoverer software, enabling comprehensive analysis of redox modifications at the peptide and protein levels.</p><p>Differential expression analysis was conducted using the MSstatsTMT package to identify proteins exhibiting significant redox changes. These proteins were further subjected to pathway and network analysis using STRING (for protein–protein interaction (PPI) and Gene Ontology (GO) enrichment) and WebGestalt (for KEGG and GSEA pathway enrichment).</p><p>Lipidomic and metabolomic analyses were also performed to explore ferroptosis-related metabolic alterations. LipidSearch was used for lipid identification and quantification, followed by statistical analysis in MetaboAnalyst. For metabolites, Compound Discoverer was used for identification and differential analysis of metabolites, with manual curation to improve accuracy. Pathway enrichment was then performed using WebGestalt. Results: Redox proteomics identified 45 redox-modified proteins associated with 79 oxidized cysteine sites, of which 5 oxidation sites were newly discovered in this study. Furthermore, two newly discovered redox-regulated proteins, PPP1R9B and S100A16, were identified. Among these 45 proteins, MDH2 and PRDX3, have been both previously reported to be associated with ferroptosis. Pathway enrichment analysis revealed a high involvement of endoplasmic reticulum (ER)-related processes, such as protein folding and ER lumen organization. PPI network analysis further identified 15 highly interconnected hub proteins, including CALR, HSPA9, PDIA3, HSP90B1, and HSPD1. Most of these ER-associated may serve as key mediators of redox regulation during ferroptosis. Lipidomic profiling identified 67 significantly altered lipid species, among which CerP(d15:0/2:0), PE(16:0e/8:0), PIP3(37:3/13:1), PE(8:0p/10:0), PIP2(16:1/21:6), PIP2(37:0/19:0), and PIP3(37:1/13:1) showed potential as ferroptosis-related biomarkers. Metabolomic analysis identified 27 dysregulated metabolites, including L-arginine, spermine, spermidine, and N1-acetylspermidine, which were significantly changed under ion-induced oxidative stress.</p><p>Integrated pathway analysis of lipidomic and metabolomic datasets showed strong convergence on L-arginine and polyamine metabolism pathways. Additionally, HIF-1 signaling was implicated as a potential regulatory axis in ferroptosis within CRC cells. In summary, this study provides a comprehensive multi-omics landscape of iron-induced ferroptosis in HCT-116 cells, integrating redox proteomic, lipidomic, and metabolomic underlying ferroptosis initiation and execution, and to uncover novel ferroptosis-related molecular candidates such as CALR, CerP(d15:0/2:0), and ether phospholipids.</p>