Liver cancer is challenging to detect in its early stages, and the global incidence rate and mortality associated with this disease have reached alarming levels. Currently, treatment options for liver cancer are limited, and there is a significant lack of safe and effective therapeutic agents. Esculetin is a natural product, exhibits almost non-toxic and inhibitory properties against various malignancies, making it a subject worthy of further investigation in liver cancer.

In this study, potential targets of esculetin in liver cancer were identified through transcriptomics, network pharmacology, and molecular docking technologies, and gene interference. Direct binding targets of esculetin were identified using surface plasmon resonance (SPR). The molecular mechanisms by which esculetin affects glucose metabolism in liver cancer were also explored. Finally, the activity against liver cancer and mechanisms of action of esculetin were validated in vivo using a mouse tumor model.

Glucose-6-phosphate isomerase (GPI) was shown to have a direct binding affinity for this compound. Esculetin inhibits glycolysis in liver cancer through its interaction with GPI and it was shown to exert a significant inhibitory effect on the genes and proteins associated with glycolysis such as ALDOA, ENO1, GAPDH, LDHA, PFKL, PGAM1, PGK1, and PKM2. Furthermore, esculetin not only suppresses the growth of liver cancer cells in vitro but also exhibits notable anti-tumor effects in vivo.

This study demonstrated the inhibitory effects of esculetin against liver cancer both in vitro and in vivo, demonstrating inhibition of glycolysis in liver cancer cells. In addition, the key glycolysis enzyme GPI was identified as a direct target of esculetin.