Ginsenoside Rh2 Regulates the Calcium/ROS/CK1α/MLKL Pathway to Promote Premature Eryptosis and Hemolysis in Red Blood Cells

Ginsenoside Rh2 (GRh2) exhibits significant potential as an anticancer agent; however, progress in developing chemotherapeutic drugs is impeded by their toxicity toward off-target tissues. Specifically, anemia caused by chemotherapy is a debilitating side effect and can be caused by red blood cell (RBC) hemolysis and eryptosis. Cells were exposed to GRh2 in the antitumor range and hemolytic and eryptotic markers were examined under different experimental conditions using photometric and cytofluorimetric methods. GRh2 caused Ca2+-independent, concentration-responsive hemolysis in addition to disrupted ion trafficking with K+ and Cl- leakage. Significant increases in cells positive for annexin-V-fluorescein isothiocyanate, Fluo4, and 2,7-dichlorofluorescein were noted upon GRh2 treatment coupled with a decrease in forward scatter and acetylcholinesterase activity. Importantly, the cytotoxic effects of GRh2 were mitigated by ascorbic acid and by blocking casein kinase 1 alpha (CK1 alpha) and mixed lineage kinase domain-like (MLKL) signaling. In contrast, Ca2+ omission, inhibition of KCl efflux, and isosmotic sucrose aggravated GRh2-induced RBC death. In whole blood, GRh2 selectively targeted reticulocytes and lymphocytes. Altogether, this study identified novel mechanisms underlying GRh2-induced RBC death involving Ca2+ buildup, loss of membrane phospholipid asymmetry and cellular volume, anticholinesterase activity, and oxidative stress. These findings shed light on the hematologic toxicity of GRh2 which is crucial for optimizing its utilization in cancer treatment.