Oridonin induces ferroptosis by inhibiting gamma‐glutamyl cycle in TE1 cells

Oridonin (Ori) is a natural tetracyclic diterpenoid active compound with excellent antitumor activity, but the mechanism of Ori on esophageal cancer cell, TE1, remains unclear. In this study, we examined the levels of intracellular iron, malondialdehyde, and reactive oxygen species after Ori treatment, while interfering with the effects of Ori with ferroptosis inhibitor, demonstrating that Ori's inhibition of TE1 cell proliferation is associated with ferroptosis. To understand the molecular mechanism of Ori, we performed UPLC–MS/MS metabolomics profiling on TE1 cells, which show that gamma‐glutamyl amino acids (gamma‐glutamylleucine, gamma‐glutamylvaline), 5‐oxoproline, glutamate, GSH, and GSSG are changed significantly after Ori treatment. Meanwhile, the activity of gamma‐glutamyl transpeptidase 1 (GGT1) decreased. This revealed that Ori inhibited the gamma‐glutamyl cycle in TE1 cells. Furthermore, we found that Ori can covalently bind to cysteine to form the conjugate oridonin‐cysteine (Ori‐Cys), resulting in the inhibition of glutathione synthesis, which is consistent with the decrease in the enzymatic activity of glutamate cysteine ligase catalytic subunit (GCLC). Eventually, the value of intracellular GSH/GSSG was reduced, and the enzymatic activity of the glutathione peroxidase 4 (GPX4) was significantly decreased. In conclusion, our experiments indicated that Ori can inhibit the gamma‐glutamyl cycle, thereby inducing ferroptosis to exert anti‐cancer activity.