Enocyanin Synergistically Enhances Sorafenib Sensitivity in Hepatocellular Carcinoma via Ferroptosis Induction Associated with p62/Keap1/Nrf2/HO-1 Pathway Inhibition

Hepatocellular carcinoma (HCC) poses a critical threat to global health because of the scarcity of effective therapeutic approaches. Sorafenib, a first-line treatment for advanced HCC, often faces efficacy limitations due to acquired resistance. Therefore, it is urgent to explore novel and effective anti-cancer drugs and combination therapies. This study explored the anti-HCC potential of Enocyanin (Eno), a natural anthocyanin-rich extract derived from grapes, either alone or combined with sorafenib. Our findings indicated that 100 μg/mL Eno remarkably suppressed the proliferation, invasion and migration of HepG2 cells, which was related to the induction of Ferroptosis characterized by increased intracellular Fe²⁺, lipid peroxidation (LPO) and Acyl-CoA synthetase long chain family member 4 (ACSL4) levels, coupled with decreased glutathione (GSH) and Glutathione Peroxidase 4 (GPX4). Mechanistically, Eno promoted Ferroptosis which was associated with inhibition of the p62/Keap1/Nrf2/HO-1 signaling pathway. Notably, Eno (100 μg/mL) combined with sorafenib (2 μM) had a synergistic anti-tumor effect (Q = 1.47), which further enhanced the inhibition of HepG2 cell growth and metastasis, aggravated Ferroptosis, and more strongly suppressed the p62/Keap1/Nrf2/HO-1 axis. In the C57BL/6 mouse subcutaneous HCC transplantation model, the combination of Eno and sorafenib showed a stronger inhibitory effect on tumor growth, reaching a 70% inhibition rate, compared to 33% with Eno alone and 55% with sorafenib alone. In summary, this study demonstrates that Eno may be a novel inducer of Ferroptosis, and it has the potential to be used in the treatment of hepatocellular carcinoma. It also provides a potential combined treatment strategy for enhancing the sensitivity of sorafenib.