Arctigenin-induced erythrocyte membrane remodeling is mediated through calcium influx, metabolic collapse, and casein kinase 1α

1. Conclusive evidence suggests that arctigenin (AGN) holds great promise in anticancer therapy but a common and poorly understood complication of chemotherapy is anemia which is precipitated by eryptosis and hemolysis. This study examines the cytotoxicity of AGN in RBCs.2. Eryptosis markers including intracellular calcium, phosphatidylserine (PS) externalization, and cell shrinkage were detected by flow cytometry using Fluo4/AM, annexin-V-FITC, and forward light scatter, respectively. Membrane blebbing was examined using electron microscopy, and photometric and potentiometric methods were used to assay hemolytic markers including hemoglobin, potassium, AST, and LDH.3. AGN significantly increased Fluo4- and annexin-V-positive cells and decreased forward light scatter which was associated with membrane blebs. While PS externalization and cell shrinkage were inhibited by extracellular calcium exclusion, suppression of hemolysis required both calcium exclusion and restriction of potassium efflux. Moreover, sucrose and mannitol rescued the cells from hemolysis while exacerbating PS externalization, which was rather significantly blunted by guanosine and CK1α inhibitor D4476.4. AGN promotes calcium-dependent eryptosis through energy depletion and CK1α activation, and exhibits a potent hemolytic activity through dysregulated ion trafficking and osmotic stress. These findings underscore the hematological toxicity of AGN and identify potential inhibitors which inform future animal studies and clinical trials.