Rupestonic acid targets ENO1 to exert antitumor activity and synergizes with paclitaxel in hepatocellular carcinoma

Rupestonic acid, a sesquiterpene, has protective properties against liver damage, inflammation, and tumor formation. Despite these known effects, its specific role and mechanism of action in combating hepatocellular carcinoma (HCC) remain insufficiently understood. This study aimed to investigate the anti-HCC effects of rupestonic acid and to identify its potential molecular targets. We employed cell counting kit-8 (CCK-8), colony formation, and flow cytometry assays to assess its impact on cell viability, proliferation, and apoptosis in HCC cell lines. Additionally, target fishing, cellular thermal shift assays (CETSA), ribonucleic acid interference, and Western blot (WB) were employed to identify rupestonic acid's protein targets in HCC cells. Our results showed that rupestonic acid significantly inhibited HCC cell proliferation, induced G0/G1 phase cell cycle arrest, and promoted apoptosis through the mitochondrial pathway. Target engagement studies employing an alkyne-rupestonic acid probe combined with mass spectrometry identified enolase 1 (ENO1) as a direct binding target, with CETSA confirming its destabilization. Furthermore, rupestonic acid inhibited the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/Forkhead box protein O (FOXO) signaling pathway, and rupestonic acid demonstrated a synergistic cytotoxic effect with paclitaxel (PTX). These findings suggest that rupestonic acid is a promising candidate for HCC treatment. They also underscore the potential of rupestonic acid in the design and development of lead compounds for HCC treatment and identify ENO1 as a viable therapeutic target.