The Mechanism Study of Etoposide Related Secondary Leukemia: Myeloperoxidase‐Catalyzed Formation of Etoposide Ortho‐Quinone Glutathione Adduct in HL60 Cells

Abstract

Etoposide is a widely used antineoplastic agent in the treatment of leukemia, but its use is associated with the increased risk of development of secondary acute leukemia. We hypothesize that one of the etoposide intermediate metabolites, etoposide ortho-quinone, can react with glutathione to form the GSH adduct in HL60 cells and therefore increase oxidative stress which may lead to DNA breaks. Myeloperoxidase (MPO)-expressing human myeloid leukemia HL60 cells were treated with etoposide in the presence and absence of a cosubstrate of MPO, hydrogen peroxide. Cell lysates and medium were analyzed by LC-ESI-ion trap-MS and MS/MS, which yielded clear evidence of the intracellular formation of the etoposide ortho-quinone-glutathione adduct. A stable isotope-labeled form of the GSH adduct was synthesized and employed as an isotope dilution internal standard in LC-ESI-quadrupole-MS analyses. The glutathione adduct formation was dependent on the concentration of etoposide. The formation of the glutathione adduct was significantly suppressed by pretreatment of HL60 with the heme synthesis inhibitor succinylacetone (p < 0.001), consistent with the idea that MPO is responsible for the conversion of etoposide to its ortho-quinone in cells. This research was supported by NIH Grant R01 CA090787