O(2)(-) is an important mediator of 2-ME-induced apoptosis. The increased oxidative stress in cancer cells forces these cells to rely more on antioxidant enzymes such as SOD for O(2)(-) elimination, thus making the malignant cells more vulnerable to SOD inhibition than normal cells.

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 radicalmediated 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. Introduction 2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17␤-estradiol that is present in human urine and blood. 2-ME is produced by sequential 2-hydroxylation and O-methylation. 1 This metabolic modification causes a loss of its ability to bind the estrogen receptor. 2-ME has been shown to have potent tumorinhibiting effects in a number of cancer cell lines in vitro and in tumor xenograft models in vivo. [2][3][4][5][6][7][8][9] This compound is currently in clinical trials, as a single agent or in combination with other anticancer agents, for treatment of several types of human cancer, including breast cancer, prostate cancer, and multiple myeloma. 2-ME also shows antileukemia activity in vitro with therapeutic selectivity. 10

Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide (O 2 . ) radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I.