Switching Death Pathways in AML; Induction of Necrosis by Arsenic, Ascorbic Acid and Antabuse (AAA).

Virtually all chemotherapy drugs induce apoptosis. Cancer cells have multiple mechanisms of resistance to this form of cell death. Necrosis may have fewer mechanisms of resistance, providing a rationale to discover ways to switch cell death pathways from apoptosis to necrosis. Arsenic trioxide (ATO) is clinically effective against acute promyelocytic leukemia and studies have suggested efficacy in treating other malignancies, including other leukemias. ATO increases the intracellular concentration of reactive oxygen species and inhibits glutathione (GSH) reduction resulting in redox changes which activate caspases and induce apoptosis. Ascorbic acid (AA) and other agents which reduce glutathione concentrations and increase hydrogen peroxide concentrations have been shown to enhance the antitumor activity of ATO in vitro. This combination of agents is currently in clinical trials. In previous experiments using pancreatic cancer cell lines, disulfiram (DSF), in clinical use as an inhibitor of aldehyde dehydrogenasel, was added to the ATO/AA combination. Synergistic induction of cell death occurred. We sought to test this combination in myeloid cell lines. We tested the ATO/AA/DSF (AAA) combination ([ATO], 1uM; [AA], 100uM; [DSF], 0.125 uM) on two myeloid leukemia cell lines, KG1 and K562. After treatment for 48 hours in AAA, annexin and propidium iodide staining revealed an increase in annexin(+) PI(+) cells (necrosis) in the KG1 cell line that was similar to the effect in pancreatic cancer cells. Nearly five fold more KG1 cells underwent necrosis after exposure to AAA than with arsenic alone. Arsenic combined with ascorbic acid or disulfiram yielded an approximately two fold increase in necrosis. We propose that the mechanism of action of disulfiram is through quenching peroxide (H2O2) radicals generated by ATO and AA. The reducion of DSF leads to oxidation of GSH and NADH which decrease [ATP]i, shifting the cells from apoptosis to necrosis. K562 cells were largely resistant to the triple combination, remaining in a state of cytostasis after exposure to AAA. BCR-ABL positive cell lines, such as K562, have a relative increase in intracellular ROS. The lack of necrosis in K562 was surprising, and suggests that the activated BCR-ABL kinase may override this proposed mechanism. Imatinib has been shown to induce necrosis, and combinations including imatinib will be tested in BCR-ABL positive cell lines. To study the potential safety of this combination for human trials, AAA was tested on peripheral blood stem cells from normal donors (obtained after informed consent) and did not appear to have synergistic effects on colony formation as compared to the drugs applied individually. These results suggest a new mechanism of cell death, switching from apoptosis to necrosis, that can be targeted to treat leukemias and other malignancies. More studies to confirm the mechanism of this cell death are underway.