Nutrient Restriction Improves the Therapeutic Efficacy of Sorafenib by Inducing Ferroptosis via the NRF2/HO-1/GPX4 Pathway in Hepatocellular Carcinoma

Background and Aims: Hepatocellular carcinoma (HCC) is a prevalent malignant tumor with a high fatality rate, making it imperative to explore novel therapeutic approaches. This study aimed to assess the effectiveness and underlying mechanism of nutrient restriction in potentiating Sorafenib-induced cell death. Methods: Cell viability was measured using MTT assays. Mitochondrial membrane potential (MMP) was assessed by the JC-1 probes. The levels of Reactive oxygen species (ROS) were determined using a DCFH-DA probe. Lipid peroxidation was quantified using the C11-BODIPY probe and a malondialdehyde (MDA) kit. Intercellular Fe 2+ was assessed using the FerroOrange probe. Western blot, HE staining, and immunohistochemistry (IHC) techniques were utilized to analyze the impact of combination therapy on NRF2, HO-1, and GPX4 proteins. Nude mice xenograft models were established to evaluate the inhibitory effects of the combination therapy in vivo. Results: Nutrient restriction/Intermittent fasting enhanced Sorafenib-induced cell death both in vivo and in vitro by elevating ROS and MDA levels, promoting excessive lipid peroxidation, and increasing intercellular Fe 2+ accumulation. Notably, key ferroptosis-associated proteins, including NRF2, GPX4, and HO-1, were significantly down-regulated by combination treatment, while glutathione (GSH) supplementation reversed this downregulation. Conclusion: The combination of nutrient restriction and Sorafenib significantly enhanced anti-tumor efficacy both in vivo and in vitro. Mechanistically, nutrient restriction potentiated Sorafenib-induced ferroptosis via the NRF2/HO-1/GPX4 pathway in HCC cells. Keywords: ferroptosis, hepatocellular carcinoma, intermittent fasting, nutrient restriction, sorafenib