Hinokitiol targets metal and redox vulnerabilities in liver cancer

Hepatocellular carcinoma (HCC) represents one of the most fatal forms of liver cancer, which accounts for most liver cancer deaths worldwide. The current medical treatments offer limited efficacy, especially in advanced stages, so there is a clear need for alternative therapeutic strategies. In recent years, natural compounds have gained attention as options for difficult-to-treat cancers. Hinokitiol (beta-thujaplicin) is one such compound. Studies have shown that Hinokitiol possesses anti-cancer properties; interestingly, this compound is also known to bind and mobilize iron. However, the link between iron-mobilizing ability and the type of stress response or cell death pathway it activates in HCC cells remains unclear. Since HCC cells inherently carry more iron, how hinokitol interacts with iron-rich environments and what specific pathway it activates in HCC cells still needs to be elucidated. We used the liver cancer cell SK-HEP1 to characterize the effect of hinokitol. We assessed the cell responses through Western blotting, RT PCR, immunocytochemistry, and live-cell imaging to measure cell viability, lysosome activity, mitochondrial integrity, autophagy, and oxidative stress. Our results showed that the Hinokitiol markedly suppressed liver cancer growth in a time and dose-dependent manner with an IC 50 ~5 µM at 96 hours; and concentrations at or above this level almost abolished clonogenic growth. At doses near IC₅₀, Hinokitiol enhanced autophagy and stress-related gene expression by roughly 2-4 fold. Western blot analysis also confirmed an increase in LC3 lipidation and lysosome markers, suggesting that Hinokitiol engages the autophagy-lysosome system rather than simple growth arrest. At the same concentrations, Hinokitiol induced the stress-response gene CHAC1 and decreased ferritin protein levels. The live-cell imaging results demonstrated that Hinokitiol treatment caused an increase in lipid peroxidation and expanded labile Fe²⁺ pool sizes in cells compared to control cells. The combined induction of CHAC1, loss of ferritin, and increased lipid damage suggest that Hinokitiol produces iron mediated oxidative stress. Taken together, these findings suggest that Hinokitiol causes cell death through an iron and lipid oxidation-driven mechanism that is independent of apoptosis and necroptosis, and is compatible with a ferroptosis-like form of cell death. By mobilizing excess intracellular iron, Hinokitiol activates ferroptosis-related pathways and takes advantage of iron and redox weaknesses in HCC cells, supporting its potential as a therapeutic agent.