Antiproliferative Wnt inhibitor wogonin prevents eryptosis following ionophoric challenge, hyperosmotic shock, oxidative stress, and metabolic deprivation

Anemia is a common complication of chemotherapy and may arise due to premature or suicidal death of red blood cells (RBCs). Prevention of RBC death thus lends itself as a promising strategy to counteract anemia. Wogonin (WGN; 5,7-dihydroxy-8-methoxyflavone) is a Wnt inhibitor derived from Scutellaria baicalensis plant with potent cytotoxic and antitumor potential. However, the nature of interaction of WGN with human RBCs is unknown. RBCs from healthy participants were exposed to different hemolytic and eryptotic stimuli for 24 or 48 hr at 37°C in the presence and absence of 100 μM WGN. Calcium overload was induced by 2 μM ionomycin, hyperosmotic shock with excessive sucrose, oxidative stress by 2-phenethyl isothiocyanate (PEITC), and metabolic deprivation by exclusion of glucose. Hemolysis was estimated from extracellular hemoglobin, phosphatidylserine (PS) exposure by Annexin V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). While WGN did not rescue the cells from the hemolytic activity of ionomycin, it reduced PS externalization by interfering with calcium influx under both ionomycin treatment and metabolic exhaustion. WGN also significantly inhibited PS exposure upon hyperosmotic shock, but the effect was independent of calcium entry. Moreover, WGN exhibited antihemolytic and anti-eryptotic activities against PEITC without appreciable reduction in ROS levels. Altogether, WGN prevents PEITC-induced hemolysis and suppresses eryptosis due to calcium accumulation, hyperosmotic shock, oxidative stress, and metabolic exhaustion. These novel insights may open new avenues into the therapeutic application of WGN for conditions in which anemia is commonly encountered, most notably cancer. Practical applications The herbal supplement Sho-Saiko-To (Xiaochaihu-tang) is commonly prescribed to relieve symptoms of liver disease. Flavonoids from the herbal constituents of Sho-Saiko-To have demonstrated considerable anti-inflammatory, antioxidant, antimicrobial, antitumor, and immunomodulatory properties. In this work, we identify WGN, a major flavonoid in Sho-Saiko-To, as a novel inhibitor of hemolysis and eryptosis of human erythrocytes. Since inordinate erythrocyte death is a major obstacle in therapeutic drug development, our findings argue for the use of WGN as a natural alternative, either as a primary or an adjuvant drug, for a wide assortment of pathological conditions including cancer.