Abstract: Objective To investigate the effect of MACC1 on RSL3-induced ferroptosis in colorectal cancer cells and explore its molecular mechanism. Methods MACC1 expression was detected in SW620, HCT116, LOVO and RKO cells using Western blotting. The effects of different concentrations of RSL3 (an inducer of ferroptosis) or Fer-1 (an inhibitor of ferroptosis) alone, or 10 μmol/L RLS3 combined with 10 μmol/L Fer-1, on viability of SW620 cells were examined using MTT assay. The survival of SW620 cells with mRNA interference of MACC1 was analyzed following treatment with RSL3, and RT-qPCR and Western blotting were performed to detect the changes in MACC1 expressions after RSL3 treatment at different concentrations and the changes in GPX4 expression after MACC1 knockdown. Flow cytometry and laser confocal microscopy were used to analyze the changes in ROS-induced lipid peroxidation in SW620 cells after MACC1 knockdown. Results SW620 cells had the highest MACC1 expression among the 4 colorectal cancer cell lines. Treatment with RSL3 significantly inhibited the viability of SW620 cells in a dose-dependent manner, while Fer-1 did not significantly affect the survival of SW620 cells. RSL3 alone reduced SW620 cell survival by 50% (P<0.01), and the combined treatment with RSL3 and Fer-1 caused no significant changes in cell survival (P>0.05). Treatment with RSL3 concentration-dependently suppressed MACC1 expressions at both the mRNA and protein levels in SW620 cells (P<0.01). MACC1 knockdown obviously enhanced the cytotoxic effect of RSL3, inhibited the expression of GPX4, and increased ROS-induced lipid peroxidation in SW620 cells (P<0.05). Conclusion MACC1 knockdown enhances RSL3-induced ferroptosis in cultured colorectal cancer cells by inhibiting the expression of GPX4.

Key words: RAS-selective lethal 3; ferroptosis; metastasis-associated in colon cancer 1; glutathione peroxidase 4