The Proteome of Colorectal Carcinoma HCT-116 Cells Undergoing Oxidative Stress and Ferroptosis Induced by Iron Overloading

Ferroptosis is an iron-dependent form of regulated cell death that holds potential implications for cancer therapy. The occurrence of ferroptosis is due to iron-induced reactive oxygen species (ROS) accumulation that causes lipid peroxidation of the cellular membranes, leading to membrane rupture and cell death. However, the key question ‒ what level of ROS can induce ferroptosis and what are the underlying molecular mechanisms and the key molecular players involved ‒ remains to be answered. The present study is based on the hypothesis that there exists a critical threshold of ROS level in tumors that regulates the gene transcription of key regulatory genes that are responsible for redox homeostasis modulation and ferroptosis progression. The aim of the study is to reveal the ROS threshold at which ferroptosis occurs in tumors and the molecular mechanisms underlying as well as the protein markers to open up new therapeutic strategies to treat or prevent the development of the tumor. An in vitro cell model was used. Colorectal carcinoma HCT-116 cell line was cultured in the presence of a series of concentrations of ferric ammonium citrate (FAC), 0 mM (the control), 1 mM, 2mM, 5 mM, 10 mM, and 20 mM to induce various levels of ROS. Cell morphology, intracellular ROS levels, cell metabolic activity, cell proliferation, and malondialdehyde (MDA, a ferroptosis marker) levels were assessed to characterize the cells. The results showed that the intracellular ROS levels increased with the increase of FAC concentration. Low concentrations of FAC promoted cell metabolic activity, whereas high concentrations of FAC decreased cell metabolic activity but induced ferroptosis in HCT-116 cells. Morphological changes were also observed with the cells undergoing ferroptosis exhibiting rounding and detachment from the growth surface.

Proteomic analysis was conducted for the control and FAC-exposed cells at various concentrations using liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein changes in abundance. Up to 3,937 high-confidence proteins in a single LC-MS/MS analysis of the protein extract prepared from the cells treated with FAC at a specific concentration were identified, a total of 5,833 high-confidence proteins were identified from all three biological replicates of each FAC concentration condition and three LC-MS/MS technical replicates. Comparisons between the cells exposed to FAC at various concentrations versus the control group identified from 27 to 240 proteins (with a fold change of ≥ 2 or ≤ 0.5, with a p-value ≤ 0.05) that were statistically differential in abundance, some of which represent potential biomarkers warranting further investigation, including APP, APOA1, THFRSF10B, THBS1, SMPD4, KDSR, UBE3C, and WWP2. Pathway analyses of the differentially abundant proteins indicate that HCT-116 cells, in response to low ROS levels, upregulate lipid metabolism pathways to maintain membrane stability and resist ferroptosis, as evidenced by active membrane metabolic systems shown in the functional analysis. At moderate ROS levels, the cells adapt by upregulating cholesterol metabolism to preserve membrane integrity. However, at high ROS levels, there is a marked activation of ferroptosis and p53 signalling pathways, alongside a decline in sphingolipid metabolism and N-glycan biosynthesis. Functional analysis suggests that these changes are accompanied by a suppression of nuclear proteins and protein-binding functions, leading to disrupted intracellular communication and pushing the cell closer to death.

Western blotting was performed to validate the proteomics data. The Western blotting results of APP and FTH1 were consistent with the corresponding LC-MS/MS results, respectively, which successfully validated the proteomics data. Moreover, the Western blotting provided additional information of APP that a phosphorylated form of APP was detected that appeared to have played an important role in promoting ferroptosis. In addition, the gene transcription factor p53 is proposed to play a role in inhibiting ferroptosis and the key antioxidation protein GPX4 was also analysed with Western blotting to gain information of their response to ROS levels.

In summary, this thesis delineates the multifaceted response of HCT-116 cells to iron-induced oxidative stress and provides a detailed map of the proteomic changes that underlie the initiation and execution of ferroptosis. The findings offer a valuable foundation for the development of targeted therapies that leverage the ferroptosis pathway for the treatment of cancer.