Metabolic Exhaustion, p38 MAPK/Rac1 GTPase/MLKL Signaling, and Ca2+ Influx Mediate Solasodine-Triggered Eryptosis

Solasodine (SOL) is a steroidal glycoalkaloid that shows a wide range of biological effects, most notably anticancer activities. Eryptosis is the programmed cell death of erythrocytes that leads to anemia. Specifically, chemotherapy-induced anemia can be precipitated in part by eryptosis triggered by anticancer agents. We hypothesize that the cytotoxic effects of SOL observed in cancer cells are nonspecific and extend to human erythrocytes. Eryptosis following SOL exposure was detected by fluorescence-assorted cell sorting analysis in erythrocytes from healthy volunteers. Phosphatidylserine (PS) translocation and cellular volume were measured using annexin-V-FITC and forward scatter (FSC), respectively. Fluo-4/AM was used to detect cytoplasmic Ca2+ and H2DCFDA was employed to probe oxidative stress. Hemolysis was also assessed by hemoglobin leakage. In addition, different incubation media and inhibitors were tested for their potential influence on SOL activity. Exposure to SOL led to modest but significant hemolysis and was paralleled by significant PS translocation, elevated FSC, and echinocyte morphology. Energy restitution through ATP, guanosine, and adenine significantly reversed PS translocation as did the removal of extracellular Ca2+ and the dissipation of the cell membrane K+ gradient. Co-treatment of erythrocytes with SOL and SB203580, NSC 23766, necrosulfonamide, caffeine, and melatonin significantly inhibited SOL-induced PS translocation. In conclusion, SOL is a novel pro-eryptotic compound whose activity is mediated through energy exhaustion, Ca2+ influx, and cytosolic KCl depletion, and requires p38 MAPK/Rac1 GTPase/MLKL signaling. Metabolic substrates, cation channel modulators, and targeted inhibition provide protective adjuncts to improve the therapeutic index of SOL as it advances toward translational applications.