OBJECTIVE To investigate the mechanism of artesunate increasing the sensitivity of hepatocellular carcinoma (HCC) cells to sorafenib in hypoxic microenvironment.

METHODS Firstly, hypoxia-resistant HCC cell models were established. NAC (a ROS scavenger), necrostatin-1 (a necrosis inhibitor), and ferrostatin-1 (a ferroptosis inhibitor) were added to examine the effects of artesunate on the inhibition rate of HCC cells and the primary modes of cell death in hypoxic microenvironment by MTT assays. Intracellular levels of GSH, ROS, lipid peroxidation product MDA, and iron ions were measured using corresponding kits. Mitochondrial membrane potential changes were assessed using JC-1 staining. Western blot and qPCR were performed to detect the expression of ferroptosis-related proteins after the addition of artesunate.

RESULTS The sensitivity of HCC cells to sorafenib decreased under hypoxic microenvironment, but artesunate significantly enhanced this sensitivity. Further analysis revealed that artesunate promoted sorafenib-induced ferroptosis by increasing ROS levels in HCC cells(P < 0.05, P < 0.01, P < 0.001). Additionally, artesunate was found to inhibit Nrf2 mRNA and protein levels (P < 0.05, P < 0.01, P < 0.001) and downregulate HIF-1α expression(P < 0.05, P < 0.01, P < 0.001).

CONCLUSION Artesunate increases intracellular oxidative stress by inhibiting Nrf2 and HIF-1α protein levels, subsequently induces ferroptosis and enhances the sensitivity of HCC cells to sorafenib in hypoxic microenvironment.