Combine immune checkpoint indoleamine 2,3-dioxygenase 1 knockdown with ferroptosis inducer Erastin/RSL3 accelerates colorectal cancer cell ferroptosis

Objective: Colorectal cancer (CRC) is a malignancy known for its aggressive behavior and notable mortality burden. Indoleamine 2,3-dioxygenase 1 (IDO1), an immune checkpoint molecule, is markedly upregulated in CRC, and it has been implicated in the regulation of key components of the ferroptosis pathway. This study aimed to elucidate the regulatory interaction between IDO1 and ferroptosis and assess the potential therapeutic effect of combined IDO1 inhibition with ferroptosis inducers Erastin and RSL3 in CRC. Material and Methods: Three CRC cell lines (CT26, MC38, and HT-29) and one normal colon epithelial cell line (NCM460) were treated with ferroptosis inducers to evaluate the changes in IDO1 expression through reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. MC38 cells with stable IDO1 knockdown were generated through lentiviral transduction. The expressions of ferroptosis-related genes following IDO1 silencing were assessed through RT-qPCR and Western blot. The antitumor efficacy of combined IDO1 knockdown and Erastin/RSL3 treatment was evaluated through cell counting kit-8, Transwell migration, and wound-healing assays. Enzyme-linked immunosorbent assay was employed to quantify intracellular levels of glutathione (GSH) and malondialdehyde (MDA). Reactive oxygen species (ROS) levels were measured via flow cytometry, and ferrous ion (Fe 2+ )and nicotinamide adenine dinucleotide phosphate (NADPH) concentrations were determined through colorimetric analysis. Results: Treatment with Erastin and RSL3 upregulated IDO1 expression in CRC cells. Silencing of IDO1 in MC38 cells resulted in increased expressions of cyclooxygenase-2, acyl-CoA synthetase long chain family member 4, and NADPH oxidase 1, alongside decreased expression of GSH peroxidase 4, solute carrier family 7 member 11, ferritin heavy chain 1, and nuclear factor E2-related factor 2. The combination of IDO1 knockdown with Erastin/RSL3 treatment significantly reduced the proliferation, invasion, and migration of MC38 cells ( P < 0.001). This combined treatment also elevated intracellular MDA, ROS, and Fe 2+ levels while lowering GSH and NADPH levels. Conclusion: Combination therapy with IDO1 inhibitor and ferroptosis inducer may be an effective way to improve treatment efficacy in CRC.