CA4P induces cell death through ROS accumulation. (A) CA4P-induced cell death can be partially prevented by coincubation with ascorbic acid, an ROS scavenger, in a concentration-dependent manner. Leukemic cells were treated with 5 nM (HL60, R81, or U937) or 10 nM (KG1a or THP-1) for 48 hours with the ascorbic acid concentrations indicated and viable cells determined by trypan blue exclusion. Results of 3 experiments in triplicate are expressed as percentage of viable cells compared with control plus or minus SEM (*P < .05 compared with CA4P-untreated cells; n = 3). (B) Early ROS accumulation during CA4P treatment. ROS levels as measured by H2DCFDA fluorescence. Numbers reflect relative mean fluorescence intensity ratio between KG1a AML cells treated with CA4P and control cells (medium alone). Results are shown from triplicate measurements (± SEM) and are representative of 3 independent experiments (*P < .05 compared with untreated cells; n = 3). (C) CA4P-mediated apoptosis of leukemic cells is reversed by inhibiting ROS and caspase pathways. HL-60 cells were treated with or without CA4P for 48 hours in presence of ROS scavenger deferoxamine (DFX) and caspase inhibitor Q-VD and the percentage of apoptotic leukemic cells was determined by annexin-V and PI staining using flow cytometry. Results are average of 3 independent experiments (*P = .01 compared with CA4P-treated cells; **P = .04 compared with CA4P+ Q-VD–treated cells).
