Figure 6. NQO1 catalyzes NAD(P)H-dependent KP372-1 redox cycling and promotes cancer cell death.

(A) Overexpression of NQO1, but not other NAD(P)H oxidases, increases KP372-1-induced NADH oxidation, H₂O₂ production, thiol oxidation, and cell death in H1299 cells. (B) The NQO1 inhibitor dicumarol, but not other pharmacologic inhibitors of ROS-generating enzymes, decrease KP372-1-induced NADH oxidation, H₂O₂ production, thiol oxidation, and cell death in A549 cells. For A and B, after cells were treated with KP372-1, SoNar fluorescence was measured immediately, while Hyper and roGFP1 fluorescence were measured after 10 min. (C-F) Knockout of NQO1 decreased KP372-1-induced cell death (C), NADH oxidation measured by SoNar fluorescence (D), H₂O₂ production measured by Hyper fluorescence (E), and thiol oxidation measured by roGFP1 fluorescence (F) in A549 cells. (G) The effect of NQO1 inhibitor dicumarol or NQO1 knockout on KP372-1-induced phosphorylation of Akt in A549 cells. Cells were treated with different concentrations of KP372-1 for 10 min. (H) Phosphorylation of Akt in different cells treated with 0.5 μM KP372-1 at the indicated times. (I) The effect of NQO1 inhibitor dicumarol on KP372-1-induced cell death. Cells were exposed to a 4-h pulse of KP372-1 either with or without 50 μM dicumarol and then grown for 24 h. (J-O) The effects of KP372-1 on tumor growth of WT A549 (J and K), NQO1 knockout A549 (L and M) and NQO1 knockout H1299 (N and O) xenografts. Tumor-growth curves (J, L and N) were measured in untreated or KP372-1-treated mice, error bars denote SEM (n=8 in each group). Xenografts were dissected at day 38 (K and M) or day 28 (O), photographed (left) and weighted (right). Error bars indicate SEM. (P) NQO1-catalyzed NADH oxidation in the presence of different concentrations of KP372-1. NADH was measured by its endogenous fluorescence. The concentrations of KP372-1 are indicated adjacent to each curve. (Q) Oxygen consumption of NADH solution (600 μM) in the presence or absence of 1 μg/ml NQO1 and different concentrations of KP372-1. Oxygen levels were measured with a BD Oxygen Biosensor System. (R) Superoxide production by a lucigenin chemiluminescence assay in an assay solution containing 600 μM NADH and KP372-1, in the presence or absence of 1 μg/ml NQO1 and/or 30 U/ml superoxide dismutase. (S) Hypothetical model for KP372-1-mediated redox cycling. Error bars represent SEM. See also Figure S6.