Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents

2-Methoxyestradiol (2-ME), a new anticancer agent currently in clinical trials, has been demonstrated to inhibit superoxide dismutase (SOD) and to induce apoptosis in leukemia cells through a free radical-mediated mechanism. Because the accumulation of superoxide (O-2(-)) by inhibition of SOD depends on the cellular generation of O-2(-) we hypothesized that the endogenous production of superoxide may be a critical factor that affects the antileukemia activity of 2-ME. In the present study, we investigated the relationship between cellular O-2(-) contents and the cytotoxic activity of 2-ME in primary leukemia cells from 50 patients with chronic lymphocytic leukemia (CLL). Quantitation of O-2(-) revealed that the basal cellular O-2(-) contents are heterogeneous among patients with CLL. The O-2(-) levels were significantly higher in CLL cells from patients with prior chemotherapy. CLL cells with higher basal O-2(-) contents were more sensitive to 2-ME in vitro than those with lower O-2(-) contents. There was a significant correlation between the 2-ME-induced O-2(-) increase and the loss of cell viability. Importantly, addition of arsenic trioxide, a compound capable of causing reactive oxygen species (ROS) generation, significantly enhanced the activity of 2-ME, even in the CLL cells that were resistant to 2-ME alone. These results suggest that the cellular generation of O-2(-) plays an important role in the cytotoxic action of 2-ME and that it is possible to use exogenous ROS-producing agents such as arsenic trioxide in combination with 2-ME to enhance the antileukemia activity and to overcome drug resistance. Such a combination strategy may have potential clinical applications.