A key novel finding in our studies is that combination of AA with the cytostatic glycolysis inhibitor synergistically induced cell death in all three NSCLC cell lines tested. In H1299 cells, the addition of 10 mM 3-PO with AA reduced the 24 h IC50 for AA by 3-fold. Similar strong synergistic interactions were also observed in the H661 and A549 cell lines. In contrast, the combination treatment caused only a 30% loss of viability in the BEAS-2B lung epithelial cell line and the DI values for the combination treatments indicate an additive effect. These results suggest that the synergistic induction of cell death induced by combinations of AA and 3-PO is selective for the NSCLC cells relative to lung epithelial cells and provide a preliminary indication that a similar combination treatment will not have toxicity issues in vivo. A more detailed analysis of the effects of the combination of AA and 3-PO over concentration ranges of revealed that the synergistic induction of cell death could be obtained by increasing the concentration of either compound relative to the other over these concentration ranges. Most combinations had CI values consistent with synergism with several combinations showing strong synergism. Several combination treatments had CI values consistent with additive or antagonistic interaction; however, all of these combinations included either 1 mM 3-PO, 50 mM AA, or both. These concentrations of 3-PO and AA alone cause less than 5% cell death and were not significantly different from vehicle-treated controls. Thus, these CI values are unlikely to accurately reflect the ability of 3-PO and AA to interact. In addition, it is possible that at 50 mM, AA exerts its well know antioxidant effect that would be expected to promote cell growth. Taken together, these results confirm and extend previous studies that demonstrate the ability of AA to enhance the activity of other anticancer compounds. AA has been reported to enhance the anticancer activity of doxorubicin, cisplatin and paclitaxel in human breast cancer cells with a clear synergism observed with a combination of AA and doxorubicin. More recently it has been shown the AA synergistically enhances cell death induced by gemcitabine in 8-Aminoadenine pancreatic cancer cells and that a combination of AA and 18A arginine synergistically induce apoptosis in a hepatoma cell line. Our studies represent the first example of AA synergistically increasing the anticancer activity of a glycolysis inhibitor. Initial mechanistic studies of the synergistic activation of cell death by combinations of AA and 3-PO revealed that production of ROS and H2O2 is likely required.