Antrocinol-mediated downregulation of ATG5 induces autophagy-dependent cell death and activates the unfolded protein response through PERK/CHOP signaling in lenvatinib-resistant hepatocellular carcinoma cells

Background: This study seeks to provide preclinical evidence demonstrating the potential of Antrocinol, a derivative of antrocin derived from the active compound of Antrodia cinnamomea, as a promising small-molecule drug candidate for overcoming drug-resistant hepatocellular carcinoma (HCC). Methods: We developed Lenvatinib-resistant Huh-7 and HepG2 cell lines (Huh-7/LR, HepG2/LR) to evaluate their viability and apoptotic response to Antrocinol. Autophagy-dependent cell death was assessed in Huh-7/LR cells using Z-VAD-FMK and shATG5 transfection. Key UPR pathway markers were analyzed via Western blot and qRT-PCR. An orthotopic hepatocellular carcinoma mouse model was used to confirm Antrocinol's in vivo effects. Results: Antrocinol reduced Huh-7/LR cell viability and increased apoptosis, with dose-dependent activation of caspase-3, -8, and -9. Z-VAD-FMK inhibited caspase activity but did not prevent apoptosis, indicating the presence of additional cell death mechanisms. Western blot and qRT-PCR confirmed UPR activation via upregulation of BiP/GRP78, PERK, eIF2α, ATF4, and CHOP. ATG5 knockdown abolished Antrocinol-induced cell death, confirming the role of autophagy. Combined with Lenvatinib, Antrocinol synergistically enhanced autophagy and apoptosis, inhibiting tumor growth in vitro and in vivo. Conclusion: This study provides the first evidence that Antrocinol, a novel hydroxylated derivative of antrocin, synergises with Lenvatinib to exert anti-proliferative effects on Lenvatinib-resistant HCC cells. Our results indicate that Antrocinol may enhance chemosensitivity in HCC by activating the unfolded protein response (UPR) pathway, specifically through the PERK/CHOP axis, and promoting autophagy-dependent cell death. These findings suggest that autophagy acts as a tumor-suppressive mechanism in HCC, with potential therapeutic implications for overcoming drug resistance.