The role of
reactive oxygen species (ROS) in the cytotoxicity of
N-(4-hydroxyphenyl)retinamide (4-HPR) was studied with use of the B-precursor
lymphoblastic leukemia cell line YCUB-2. The increase in intracellular ROS measured with 2'-7'-dichlorodihydrofluorescein diacetate after 3 hours' incubation was 3.7-fold with 1 microM
4-HPR and 5.8-fold with 5 microM
4-HPR. The rate of apoptosis after 48 hours' incubation was 9.8% and 56.4% in comparison with untreated cells.
Hydroethidine, which is a more specific
indicator of
superoxide anion radical level, did not effectively detect 4-HPR-induced ROS. The
antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one suppressed 4-HPR-induced ROS production and apoptosis. The cytotoxicity of
4-HPR was analyzed in 4 other
leukemia/
lymphoma lines (CCRF-HSB2, Molt-4, KG-1, HL-60). We found that the cytotoxicity of
4-HPR correlated with the amount of ROS produced in cell lines, except in HL-60 cells. The intracellular
glutathione level varied among the 5 cell lines, the highest levels occurring in Molt-4 and KG-1, which were less sensitive to
4-HPR. Suppression of
glutathione by
buthionine sulfoximine enhanced the level of 4-HPR-induced ROS production and apoptosis in Molt-4. Our findings suggest that ROS play a significant role in the antileukemia effect of
4-HPR and that the
glutathione level in
leukemias may be associated the sensitivity of the cells to
4-HPR.