Arenobufagin causes ferroptosis in human gastric cancer cells by increasing rev-erbα expression

BACKGROUND AND AIMS: Gastric cancer is the fifth most diagnosed malignant tumor worldwide with limited effective chemotherapy. Ferroptosis is a new type of programmed cell death, which is becoming as a novel therapeutic target for tumors. Arenobufagin (ArBu) is a bufadienolide isolated from toad skin and venom, which exhibits broad-spectrum anti-tumor activity. It is unclear whether ArBu causes ferroptosis, thereby exhibiting anti-tumor activity in gastric cancer. We aimed to determine whether ArBu causes ferroptosis in cultured human gastric cancer cells. EXPERIMENTAL PROCEDURE: Different human gastric cancer cells were treated with ArBu (5–20 μM, 48 h). Indicators of apoptosis and ferroptosis were measured. CRISPR/Cas-9 system was employed to delete Nr1d1 gene. RESULTS: ArBu incubation reduced cell viability in a concentration-dependent manner. ArBu caused ferroptosis but not apoptosis at a lower concentration (10 μM), despite it caused both of them at a higher concentration (20 μM). Cotreatment with a selective ferroptosis inhibitor ferrostatin-1 protected against ArBu (10 μM)-induced reduction in cell viability. ArBu-mediated ferroptosis was associated with abnormal expression of genes involved in iron uptake, lipid peroxidation, and antioxidants. Particularly, Nr1d1 gene expression was most significantly increased after ArBu treatment. Furthermore, activating Rev-erbα encoded by Nr1d1 by a selective agonist GSK4112 (1 and 2 μM, 48 h) caused ferroptosis. In contrast, Rev-erbα knockout using the CRISPR/Cas-9 system diminished ArBu-induced ferroptosis in cultured human gastric cancer cells. CONCLUSION: ArBu causes ferroptosis by increasing Rev-erbα expression in human gastric cancer cells. This has implications of ArBu as a promising therapy for gastric cancer. SECTION: 1. Natural Products; TAXONOMY (CLASSIFICATION BY EVISE): Traditional medicine, pharmacology, gastric cancer, signal pathway.