Insights in CYP2E1-mediated ferroptosis in hepatocellular carcinoma

Introduction: CYP2E1, a cytochrome P450 enzyme mainly found in hepatocytes, is crucial for metabolizing xenobiotics including fatty acids and drugs. In hepatocellular carcinoma (HCC), CYP2E1 acts as a tumor suppressor. However, CYP2E1 has significantly reduced RNA and protein expression in HCC and its expression correlates positively with patient survival. We hypothesized that CYP2E1 promotes ferroptosis-a regulated, iron-dependent cell death involving iron accumulation, ROS generation, glutathione depletion, and lipid peroxidation-which may underlie its beneficial effects in HCC. Methods: HCC cell lines were cultured per ATCC guidelines and transduced with adenoviral vectors expressing GFP or CYP2E1, confirmed by Western Blot. To assess palmitic acid (PA) toxicity, cells were treated with 0-5mM PA for 24 and 48h, followed by cell death analysis. For ferroptosis sensitivity, cells were exposed to graded concentrations of inducers (FINO2, FIN56, Erastin, RSL3) for 24h, then analyzed for cell death. Results: Cell lines exhibited a variation in sensitivity to palmitic acid (PA) treatment, with HepG2 exhibiting a stronger response than SNU398 and SNU449 cells. However, there was no statistically significant effect of CYP2E1 expression in the response to PA feeding. Sensitivity to ferroptosis inducers also varied widely across cell lines and appeared to be cell type-specific rather than dependent on CYP2E1 expression. Conclusion: Our results provide new insight into the sensitivity of different HCC cell lines to ferroptosis induced by PA feeding and by pharmacological inducers of ferroptosis. Further studies are needed to determine the mechanism of CYP2E1 tumor suppression, which our data suggests may not be through ferroptosis.