Inhibition of Suicidal Erythrocyte Death by Indirubin-3’-Monoxime

Abstract

Background/Aims: Qing Dai is a prized traditional Chinese medicine whose major component, indirubin, and its derivative, indirubin-3’-monoxime (IDM), have inhibitory effects on the growth of many human tumor cells and pronounced anti-leukemic activities. However, the effects of IDM on mature human erythrocytes are unclear. This study aimed to evaluate the potential impact of IDM on erythrocytes and the mechanisms underlying that impact. Methods: Utilizing flow cytometry and confocal laser scanning microscopy, phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate (FITC). The relative cell size, expressed in arbitrary units, was evaluated by forward scatter in a flow cytometer. Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca2+ activity, reactive oxygen species (ROS) formation was assessed by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence, and ceramide abundance was evaluated by FITC-conjugated specific antibodies. Results: The 24-h exposure of human erythrocytes to IDM (12 µM) significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration ([Ca2+]i). IDM (3-12 µM) did not significantly modify the ceramide level or DCFH-DA fluorescence. Energy depletion (removal of glucose for 24 hours) significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter, and these effects were significantly mitigated by IDM (12 µM). Moreover, the Ca2+ ionophore ionomycin (1 µM, 60 min) and oxidative stress (30 min exposure to 0.05 mM tert-butyl hydroperoxide, t-BHP) similarly triggered eryptosis, which was also significantly suppressed by IDM. Conclusions: IDM is a novel inhibitor of suicidal erythrocyte death following ionomycin treatment, t-BHP treatment and energy depletion. Thus, IDM may counteract anemia and impairment of microcirculation, at least in part, by inhibition of Ca2+ entry into erythrocytes.