TSA as an NQO1 Specific Target Agent Figure 4. TSA induces NQO1 dependent generation of ROS in NSCLC cells. Intracellular ROS generation measured by DCF staining. A, for time course study, cells were treated with 10 mM of TSA with or without pretreatment by 10 mM of DIC or 5 mM of NAC; B, cells were exposed to indicated doses of TSA for 1 h; C, effect of NQO1 silence on TSA triggered ROS formation; cells were exposed to 4 mM or 40 mM of TSA for 1 h; D, H595-NQO1 cells and H596-vector cells were exposed to 10 mM or 40 mM of TSA for 1 h. H2O2 (400 mM) was used as a positive control. E and F, Glutathione cycling assays; E, NSCLC cells were exposed to indicated doses of TSA with or without DIC pretreatment for 24 h. F, H596-vector and H596-NQO1 cells were exposed to TSA for 24 h. Data are shown as relative changes to blank controls and expressed as mean 6 SE of at least three independent experiments. (# P,0.05, ## P,0.01, ### P,0.001 NQO1 siRNA vs. control siRNA, H596-NQO1 vs. H596-vector, A549 with DIC vs. TSA alone; * P,0.05, ** P,0.01, *** P,0.001, TSA or H2O2 treatment vs. their respective control cells). doi:10.1371/journal.pone.0042138.g004 of TSA was further confirmed in the A549 tumor xenografts. TSA (20 mg/kg) treatment significantly retarded the tumor growth, whereas the co-administration of DIC could largely abrogate this effect. It was very interesting to note that DIC significantly increases the tumor accumulation of TSA while reduces its anti- tumor efficacy (Fig. 8). This can be explained by the fact that TSA was predominately metabolized by NQO1 and UGTs. However, NQO1 had little influence on the cellular uptake of TSA, as evidenced from the observation that the cellular accumulations of TSA in A549 and H596 cell lines was almost identical and the siRNA silence of NQO1 in A549 cells had little effect on TSA uptake (Fig. S2). Such a pharmacokinetic and pharmacodynamic paradox strongly indicates that the NQO1 mediated bioactivation is pivotal on determining TSA’s anti-NSCLC efficacy. The major characteristics of NQO1 catalyzed bioactivation is to produce a highly unstable catechol intermediate that auto-oxidizes to the parent quinone forming a futile redox cycle which renders cells to continuous ROS challenges. It has been recently reported that ROS produced from NQO1-mediated redox cycle played crucial role in Lap induced apoptosis [40]. It seems that ROS may also play an important role in TSA induced cell death, since NAC co-treatment could largely abolish TSA induced DNA damage, cell apoptosis, and cytotoxicity in NQO1 positive cells (Fig. 1, 2, and 3). The p53 tumor suppressor is a pivotal regulating component that senses various intrinsic and extrinsic stresses and initiates apoptotic cell death [41]. Moreover, p53 protein degradation is negatively regulated by NQO1 via the inhibition of an ubiquitin- independent 20S proteosome mediated degradation pathway [42]. Therefore, it is important to determine the role of p53 on the apoptotic cell death induced by NQO1 substrates. All evidence collected from the present study indicated that TSA induced apoptotic cell death is p53 independent. First, the pretreatment with typical p53 inhibitor and the p53 silence by siRNA had negligible effect on TSA induced cytotoxicity, apoptosis, and all the tested events of mitochondrial apoptotic pathways in A549 cells (Fig. 1F, Fig. 2A, B, Fig. 5, Fig. 6, Fig. 7). Second, TSA could induce apparent and concentration dependent cytotoxicity and apoptosis in H596-NQO1 cells (Fig. 1E, 2C) in which the p53 is well known to be mutated [43]. Finally, TSA treatment in A549 cells induced a concentration dependent effect on decreasing p53 protein contents, and of interest, the NQO1 inhibitor DIC can synergize with TSA on decreasing p53 protein content in A549 cells but reverse the downregulation of NQO1 protein levels (Fig. S3). Although the detailed mechanisms underlying TSA’s effect on PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e42138