TSA as an NQO1 Specific Target Agent Figure 3. TSA induces NQO1-dependent and ROS mediated DNA damage. DNA lesions determined by alkaline comet assays. A, TSA induced DNA damage in A549 and H596 cells, as well as the effect of NAC or DIC pretreatment. B, effect of NQO1 silence in TSA induced DNA damage in A549 cells. C, DNA damage in H596-NQO1 and H596-vector cells. Left panel, the representative morphological appearance of cells with TSA treatment for 24 h; right panel, quantitative results expressed as fold change (T/C) of comet tail lengths or expressed as comet tail lengths (arbitrary unit). Data are shown as mean 6 SE of at least three independent experiments. (# P,0.05, ## P,0.01 NQO1 silence vs. control siRNA or H596- NQO1 vs. H596-vector; * P,0.05, ** P,0.01, *** P,0.001, TSA or H2O2 treatment compared with control cells). doi:10.1371/journal.pone.0042138.g003 To assess the tumor accumulation levels of TSA, tissue distribution study was performed in mice bearing A549 tumor xenografts (Fig. 8C). Consistent with our previous study of TSA distributions in rats [37], TSA that was prepared as a solid dispersion with PEG6000 preferentially distributed into the reticuloendothelial systems. Despite this limitation, TSA could still efficiently distribute into the tumor tissues; TSA showed higher accumulation levels and prolonged exposure times in tumors than that in plasma. DIC pretreatment, despite antagonizing the tumor suppression effect of TSA, significantly increased the exposure levels of TSA including that in tumor tissues, by a mechanism of inhibiting TSA metabolism via NQO1/UGTs [31]. Improving TSA delivery that can escape from reticuloendothelial system uptake would be expected to further increase its tumor accumu- lation levels and thus better anti-tumor efficacy. Discussion Accumulating evidence suggests that NQO1 is a promising therapeutic target for various tumors, especially for NSCLC, in which NQO1 is overexpressed compared with normal lung tissue [14]. Nevertheless, there is currently no specific NQO1 target drugs used in clinic and thus its therapeutic value remains unexplored. Although some anti-tumor drugs such as mitomycin can be bioactivated by NQO1, all of them lack sufficient selectivity and specificity to NQO1 and their anti-tumor effects are better explained by other mechanisms [38,39]. Evidence collected from the studies of Lap indicates that the NQO1 activated futile redox cycle can induce a unique cell death pathway, inspiring the new idea of developing NQO1 target anti-tumor drugs. This study contributes to identify a specific NQO1 substrate TSA, which induces a unique p53 independent activation of mitochondrial apoptotic pathway upon NQO1 bioactivation. The hypothesis of NQO1 as the intracellular target of TSA was based on previous findings that NQO1 is the rate controlling enzyme in determining TSA metabolism [31,32]. To verify it, we studied the role of NQO1 in determining the anti-cancer effects of TSA against NSCLC. In all tested aspects, TSA exhibited in an NQO1 dependent manner on inducing cytotoxicity, apoptosis, ROS production, and DNA damage; all of these events could be observed only in the NQO1 positive cell lines A549 and H596- NQO1. Consistently, the silence of NQO1 by specific inhibitor DIC and siRNA could largely abrogate all of these events in A549 cells. More importantly, the NQO1 dependent anti-cancer effect PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e42138