prognostic and predictive factor in the poor outcome of breast cancer [17]. These findings suggest that the pharmacological role of NQO1 is far beyond its enzymatic activity on reducing quinones. Taking together, it would be of high interest to determine the therapeutic potentials and underlying mechanisms of NQO1 target agents on tumors. b-Lapachone (Lap), a well studied NQO1 substrate, has been identified as a promising agent for various cancer therapy [18]. However, repeated oral treatment of Lap induces anemia in both rats and humans which may greatly limit its application [19,20]. Another limitation of Lap is its NQO1 specific activity can only be realized within a relatively narrow concentration range and exposure time window [18]; it is questionable whether such a condition can be well controlled in the in vivo states. Tanshinone IIA (TSA) was a derivative of phenanthrene- quinone isolated from Salvia miltiorrhiza (Danshen, in Chinese), a widely used herbal medicine. Although its traditional use mainly focused in cardiovascular and cerebrovascular diseases [21,22,23,24], the anti-tumor activity of TSA and other structur- ally similar tanshinones were confirmed during the past decades [25,26,27]. Various studies showed that TSA exerted cytotoxic effects against a wide range of human tumor cell lines [28]. In spite of previous efforts on disclosing its mechanisms [29,30], the molecular target of TSA to elicit its anti-tumor effect remains elusive. We have recently found that NQO1 catalyzed quinone reduction followed by immediate glucuronidation is the predom- inant metabolic pathway of TSA in both rats [31] and humans [32]. NQO1 reduces TSA to form a highly unstable catechol intermediate which auto-oxidizes back to parent TSA and constitutes a futile redox cycle producing oxidative stress [31]. These findings prompted us to hypothesize that NQO1 may be the primary intracellular target of TSA in cancer cells. The present study aims to determine whether NQO1 plays a pivotal role on initiating the anti-tumor effect of TSA both in vitro and in vivo. Isogenically matched NQO1+ and NQO12 human H596 and NQO1 high expression human A549 NSCLC cell lines were applied to determine the NQO1 dependent cytotoxicity, apoptotic effect, ROS production, DNA damage, and cellular uptake of TSA. We demonstrate that TSA is a promising NQO1 specific target agent, which induces apoptotic cell death of NSCLC cells via a unique NQO1-initiated and ROS-mediated activation of a p53-independent but caspase-dependent mitochon- drial apoptotic pathway. Results TSA induced cytotoxicity is NQO1 dependent and p53- independent To determine the role of NQO1, TSA cytotoxicity assays were performed in NQO1 high expression A549 cells, NQO1 negative and transfected H596 cells; NQO1 protein levels and enzyme activities in NSCLC cells are shown in Fig. 1A. Protein expression and enzyme activity of NQO1 could not be detected in H596 cells; the NQO1 enzyme activity in H596-NQO1 cells is much lower than that in A549 cells, which is consistent with previous findings [18]. TSA induced a concentration dependent cytotoxicity in A549 cells with an IC50 value at 5.32 mM. In contrast, NQO1 negative H596 cells were largely resistant to TSA cytotoxicity with an IC50.40 mM (Fig. 1B). NQO1 inhibition by the specific NQO1 inhibitor dicoumarol (DIC,10 mM) could largely prevent TSA’s cytotoxicity in A549 cells; NQO1 siRNA knockdown could also abrogate the cytotoxic effect of TSA, albeit to a lesser extent to that by DIC (Fig. 1C, D). The transfection of NQO1 to H596 cells restored its susceptibility to TSA induced cytotoxicity. The TSA as an NQO1 Specific Target Agent IC50 of TSA in H596-NQO1 and in H596-vector cell lines is of 23.56 mM and .80 mM, respectively (Fig. 1E). Of note, the IC50 values in A549 cells were much lower than that in H596-NQO1 cells, which is in agreement with the difference of enzyme activities between these two cell lines. All these results strongly suggest that TSA induced cytotoxicity is NQO1 dependent. In addition, it seems that TSA showed better NQO1 target specificity than Lap; the IC50 value difference between A549 cells and H596 cells for TSA (5.32 mM VS.40 mM) is much higher than that for Lap (1.94 mM VS 3.93 mM). Moreover, the pretreatment with typical p53 inhibitor pifithrin-a (PFT-a) [33] and the p53 silence by siRNA had negligible effect on TSA induced cytotoxicity (Fig. 1F), suggesting TSA induced cytotoxicity in a p53-independent manner. TSA induces NQO1 dependent and p53-independent apoptotic cell death TUNEL assays were performed to confirm that TSA induced cell death is due to the induction of NQO1 dependent apoptosis. As shown in Fig. 2A, TSA induced a concentration dependent apoptosis and the TUNEL positive cells reached over 80% upon 40 mM of TSA treatment in A549 cells, whereas the TUNEL positive cells detected from H596 cells upon TSA treatment is of no difference from control treatment. In addition, both DIC and NQO1 siRNA could significantly reverse TSA induced apoptosis in A549 cells (Fig. 2A, B). Likewise, the transfection of NQO1 to H596 cells significantly restored its susceptibility to TSA induced apoptosis, characterized with a 38.02% versus 7.28% TUNEL positive cells in H596-NQO1 cells and H596-vector cells (Fig. 2C), respectively. The results of TUNEL assays are in complete agreement with those from MTT based cytotoxicity studies, strongly suggesting that TSA induces an NQO1 dependent apoptotic cell death in NSCLC. Consistent with the MTT result, the TUNEL assay also showed that p53 inhibitor PFT-a and P53 siRNA had insignificant effect on TSA induced apoptosis, indicating a p53 independent apoptosis induced by TSA. TSA induces NQO1 dependent DNA damage Because DNA damage is a pivotal initiator on inducing apoptotic cell death [34], we sought to determine whether TSA treatment induces NQO1-dependent DNA damage and breaks. As expected, TSA induced a dramatic and concentration dependent DNA damage in A549 and H596-NQO1 cells but minimal in NQO1 negative H596 and DIC or siRNA pretreated A549 cells (Fig. 3A, B and C). H2O2 (200 mM) treatment for 4 h induced almost identical DNA damage in all the cell lines. Time course data showed that the TSA induced NQO1-dependnet DNA damage reached the maximum after 24 h treatment but slightly decreased after 48 h, possibly because of excessive cell death (Fig. 3A). It was also noted that TSA at 40 mM induced quite similar amount of DNA damage in A549 cells and H596- NQO1 cells in spite of different NQO1 enzymatic activity between A549 cells and H596-NQO1 cells. We proposed that it is possibly that TSA at 40 mM may induced a maximum DNA damage for both A549 and H596-NQO1 cells and thus no difference was observed. However, A549 cells are indeed to be much more sensitive than H596-NQO1 cells to TSA induced DNA damage at lower dose challenge (Fig. 3B, C). ROS produced from NQO1 bioactivation is an important mediator on TSA induced apoptotic cell death We have found that TSA was reduced by NQO1 to produce a highly unstable catechol intermediate which could be either PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e42138