TSA as an NQO1 Specific Target Agent Figure 6. TSA activates an NQO1-initiated, ROS-mediated, and p53- independent mitochondrial apoptotic pathway. A549 cells were exposed to TSA of gradient concentrations (4, 10, and 40 mM) alone or pretreated with DIC, NAC, or subjected to NQO1 and p53 silence, prior to the treatment of 40 mM of TSA for 48 h. A, whole cell lysates were prepared after indicated treatment and western blot analysis was conducted using anti- Bax, -Bcl-xl, -PARP, -cleaved PARP, -NQO1, -p53, and -GAPDH antibodies. B, Cytosolic and mitochondrial proteins were prepared for western blot analysis using anti-cytochrome c antibody; fluctuations in protein loading between samples were monitored by GAPDH or COX-IV levels, respectively. The relative density value of each band is shown below the Western blot. The data are representative of a typical experiment that was conducted three times (mean values, P,0.05). doi:10.1371/journal.pone.0042138.g006 independent mitochondrial apoptotic pathway initiated by NQO1 and mediated by ROS (Fig. 9). All aspects of in vitro studies indicate that TSA is a promising NQO1 target agent that may be developed to an effective therapeutic drug against NSCLC. To further validate the prospect of TSA development, the NQO1-dependent antitumor effect of TSA was investigated in vivo in A549 cells inoculated nude mice. TSA significantly inhibited the tumor growth (Fig. 8). DIC treatment alone could also partially inhibit the tumor growth, which is consistent with a previous report that DIC slowed the tumor growth and extended mice survival [47]. However, the DIC co-treatment could significantly reduce the antitumor activity of TSA. Although the antagonizing effect of DIC observed in vivo seems less strong than that observed in the in vitro studies, such a relatively weaker effect can support the NQO1 dependency of TSA in inhibiting the tumor growth, considering that DIC itself can partially retard the tumor growth and that DIC treatment increased the TSA accumulation levels in tumor tissues by inhibiting its metabolic eliminations (Fig. 8) [31]. Thus, both in vitro and in vivo data strongly support that TSA is a promising specific NQO1 target anti-tumor agent. In particular, TSA seems possess a wide therapeutic window because of its high NQO1 specificity and selectivity and its negligible toxicity in normal tissues (Fig. 8B). In the in vitro conditions, the NQO1 negative H596 cells are largely resistant to TSA induced toxicity even at a high concentration up to 80 mM and prolonged exposure times up to 72 h, which may represent a superior feature of TSA as compared with Lap (Fig. 1B) [18]. The in vivo histological examinations support that TSA has no toxic effects in normal tissues (Fig. 8). Our study showed that the NQO1 irrelevant cytotoxicity of TSA is much lower than that of Lap (Fig. 1B). TSA is a natural compound contained in Danshen, a Chinese medicine that have been widely used clinically for the therapy of various cardiovascular and cerebrovascular diseases; in addition, the TSA sodium sulphate derivative has been used in China as a cardiovascular drug for more than 20 years and no side effect of anemia has been found [48,49,50]. All lines of evidence hint to that TSA is unlikely to induce anemia that has been found for Lap; nevertheless, it is important in the future preclinical safety evaluation of TSA to monitor erythrocytes count. The major PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e42138