Inhibitory Effects of Esculetin on Liver Cancer Through Triggering NCOA4 Pathway-Mediation Ferritinophagy in vivo and in vitro

OBJECTIVE: To explore the effects of Esculetin on liver cancer and explore potential mechanisms of Esculetin-inducing cells death. METHODS: Esculetin’s effects on the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells were detected by using CCK8, crystal violet staining, wound healing, Transwell(TM) and Annexin V-FITC/PI. Flow cytometry, fluorescence staining, Western blot, T-AOC, DPPH radical scavenging assay, hydroxyl radical’s inhibitory capability and GSH test were used to examine the esculetin’s effects on the ROS level, the oxidation-related substances and proteins’ expression in hepatoma cells. In vivo experiment was performed by xenograft model. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by esculetin. Live cell probe, Western blot, Fe(2+) content, MDA, HE staining, Prussian blue staining and immunohistochemistry were used to examine the ferritinophagy-related phenomenon induced by esculetin in hepatoma cells. The relationship between esculetin and NCOA4-mediated ferritinophagy was confirmed through gene silence and overexpression, immunofluorescence staining and Western blot. RESULTS: Esculetin suppressed the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells significantly, influenced the oxidative stress level, altered the autophagy and iron metabolism levels in cells, and produced a ferritinophagy-related phenomena. Esculetin increased the levels of cellular lipid peroxidation and reactive oxygen species. In vivo, esculetin could decrease tumour volume, promote LC3 and NCOA4 expressions, suppresse hydroxyl radical‘s inhibiting capacity and GSH, increase Fe(2+) and MDA levels, decrease antioxidant proteins expression in tumour tissue. In addition, Esculetin could also increase the iron deposition of tumour tissues, promote ferritinophagy, and induce tumours’ ferroptosis. CONCLUSION: Esculetin has an inhibitory effect on liver cancer in vivo and in vitro through triggering NCOA4 pathway-mediation ferritinophagy.