PLCγ1 is required for Wogonin-induced apoptosis in malignant T cells. (A) PLCγ1def Jurkat cells are resistant to Wogonin-induced apoptosis. Jurkat deficient either in PLCγ1 (J- PLCγ1def), SLP76 (J-SLP76def), or LAT (J-LATdef) together with the retransfected J-PLCγ1retran, J-SLP76retran, and J-LATretran cells were subjected to Wogonin treatment. Apoptotic cells were determined by either a decrease in fetal calf serum/SSC (left panel) or DNA fragmentation (right panel). Results are representative of 3 or 4 independent experiments. Error bars represent SD. (B) Wogonin generates rapid and strong H₂O₂ signals in malignant T cells. Jurkat and normal (day 6) T cells were treated with Wogonin (50 μM) for the indicated times. The H₂O₂ products were monitored by oxidation-sensitive fluorescent dye by FACS. (C) Wogonin generates long-lasting H₂O₂ signals compared with αCD3 stimulation. Jurkat cells were stimulated with either Wogonin (50 μM) or αCD3 (coated at 30 μg/mL) for the different times as indicated. The H₂O₂ products were monitored by FACS. (D) Wogonin induces phosphorylation of PLCγ1. Jurkat cells were stimulated with either αCD3 (coated at 30μg/mL) or Wogonin (50 μM) for indicated times. The expression levels of activated PLCγ1 were analyzed by FACS with antiphospho-PLCγ1 antibodies. (E) Wogonin induces stronger activation of PLCγ1 in malignant than in normal T cells (P = .035, n = 4). Jurkat and normal T cells isolated from peripheral blood of healthy donors were treated with Wogonin (50 μM) for 24 hours. In parallel, T cells activated by αCD3 for 24 hours were used as a positive control to demonstrate that the T cells used were capable to express activated PLCγ1. The expression levels of phosphor-PLCγ1 were analyzed as in panel D). Error bars represent SD. (F) Schematic summary of the discovered mechanism by which Wogonin induces malignant T cells to undergo apoptosis. Tumor cells produce ROS at elevated rates and Wogonin shows stronger and sustained generation of H₂O₂ in the malignant but not in normal T cells. H₂O₂ in turn serves as a signaling molecule to activate PLCγ1 and consequently induces Ca²⁺ release from intracellular stores. Cytosolic Ca²⁺ overload leads to disrupt the mitochondrial membrane. Nifedipine, FK-506, CsA, and Ru-360 were shown to block the Wogonin-induced apoptosis pathway at different levels.