Abstract
Sorafenib is a potent inducer of ferroptosis used to manage hepatocellular carcinoma (HCC). The ferroptosis induced by sorafenib activates the p62-Keap1-Nrf2 pathway. Abnormal activation of Nrf2 reduces sorafenib's efficiency and ferroptosis action and induces sorafenib's resistance. Consequently, our study tried to study the effect of a novel combination of sorafenib and Camptothecin (CPT, Nrf2 inhibitor) to improve sorafenib's ferroptosis action and reduce sorafenib resistance in the treatment of HCC. We evaluated the efficacy of sorafenib and/or CPT using HepG2 and Huh7 cell lines. MTT assay evaluated the anti-proliferation effects. The combination index (CI) and dose reduction index (DRI) were calculated using Isobologram analysis. Malondialdehyde (MDA), total antioxidant capacity (TAC), iron concentration, glutathione peroxidase (GPX4), and glutathione reductase (GR) activity assays were used to determine the ferroptosis action of drugs. Western blot was used to investigate the expression of the implicated proteins. Bioinformatics tools were used to determine the correlation between these proteins. Finally, the HPLC technique is used to measure cellular drug uptake. Our results revealed a strong synergism between sorafenib and CPT. The synergetic combination significantly increases lipid peroxidation and iron concentration, decreases TAC, GPX4 and GR activity, and reduces the expression of both Nrf2 and SLC7A11. The downregulation of Nrf2 expression has a vital role in the reduction of resistance mediators to sorafenib against HCC cells like (p62, MT1G, and ABCG2) and improves the cellular uptake of sorafenib. The current study provided evidence that Nrf2 inhibition by CPT improves sorafenib's sensitivity and reduces sorafenib's resistance via the augmentation of sorafenib's ferroptosis action.