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

Abstract

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.