TSA as an NQO1 Specific Target Agent Figure 1. TSA induced cytotoxicity is NQO1 dependent, ROS mediated, and p53-independent (MTT assay). A, protein levels and enzyme activities of NQO1 in NSCLC cells. NQO1 activity ,1.0 nmol?min21?mg21 was indicated non-detectable (ND). B, cytotoxicity of TSA and Lap in NSCLC cells; cells were exposed to gradient concentrations of TSA (0.4–40 mM) or Lap (0.5–10 mM) for 72 h. C, effects of NAC or DIC pretreatment; A549 cells were pretreated with 5 mM of NAC for 1 h or 10 mM of DIC for 30 min. D, effects of NQO1 silence in A549 cells. E, TSA cytotoxicity in H596 and NQO1-transfected H596 cells. F, effect of P53 inhibition or silence; A549 cells were pretreated with 10 mM of PFT-a for 5 h or pretreated with P53 siRNA. Data are shown as mean 6 SE of three independent experiments. doi:10.1371/journal.pone.0042138.g001 conjugated with glucuronic acid in the presence of UDP- glucuronic acid transferases (UGTs) or auto-oxidizes back to TSA without them [31]. Since lack of UGTs was observed in NSCLC cells, it is reasonable to postulate that TSA can trigger a futile redox cycle producing excessive ROS in NQO1 positive cells. To support this consideration, DCF staining and glutathione cycling assay were applied to monitoring TSA induced ROS formation. Time course of DCF staining data showed that TSA (10 mM) induced excessive ROS formation within 30 min and reached saturation in 60 min in A549 cells but not in H596 and DIC pretreated A549 cells (Fig. 4A). NQO1 silence largely prevented TSA induced ROS formation in A549 cells (Fig. 4C). Consistently, the transfection of NQO1 to H596 cells restored the capability of TSA induced production of ROS from the futile cycle (Fig. 4D). Results obtained from the glutathione cycling assay (Fig. 4E, F) further supported that TSA induced ROS production in an NQO1 dependent manner. In contrast, H2O2 induced almost identical ROS production in all cases, independent of NQO1 expression. In view that NQO1 triggered ROS production is a very early and continuous event, it is important to determine whether ROS produced from TSA quinone and catechol recycling is a key player in TSA induced apoptotic cell death. The pretreatment of A549 cells with 5 mM N-acetyl cysteine (NAC), a typical ROS scavenger, was sufficient to remove TSA induced intracellular ROS (Fig. 4A). NAC pretreatment could significantly prevent TSA induced cytotoxicity (Fig. 1C), apoptosis (Fig. 2A), and DNA damage (Fig. 3A). Moreover, in all test aspects, the reversing effects of NAC pretreatment are almost comparable with that achieved by DIC pretreatment. All the results indicated that ROS produced from NQO1 catalyzed recycling process is an important mediator on inducing apoptotic cell death of NSCLC cells. TSA caused NQO1 and ROS-mediated, p53 independent activation of mitochondrial pathway As shown in Fig. 5, TSA caused a dose-dependent disruption of MMP, which is consistent with a previous study by Chiu et al [35], in which TSA was found to induce a ROS mediated mitochon- drial apoptotic cell death. Because we found herein that TSA induced apoptotic cell death was NQO1 dependent, we thus sought to determine the role of NQO1 in activating the mitochondrial apoptotic cell death pathway. NQO1 inhibitor DIC, NQO1 siRNA, as well as NAC pretreatment could significantly reverse TSA induced MMP disruption, indicating a NQO1 dependent and ROS-mediated mitochondrial disruption PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e42138