TSA as an NQO1 Specific Target Agent Figure 2. TSA induced apoptosis is NQO1-dependent, ROS-mediated, and p53-independent (TUNEL assay). A, upper, A549 cells and H596 cells were exposed to indicated concentration of TSA for 48 h; lower, A549 cells were pretreated with 10 mM of DIC for 30 min, 5 mM of NAC for 1 h, or 10 mM of PFT-a for 5 h and then exposed to 40 mM of TSA for 48 h. B, effects of NQO1 or p53 silence on TSA induced apoptosis in A549 cells. C, H596-vector and H596-NQO1 cells were exposed to TSA for 48 h; the TUNEL assay was performed using Click-iT TUNEL Alexa Fluor 594 Imaging Assay Kit. Eight to ten fields were examined in each experiment, the TUNEL signal is shown in red and Hoechst 33342 nuclear stain is shown in blue. Right are the representative images of TUNEL staining. All data are shown as mean 6 SE of three independent experiments (# P,0.05, ## P,0.01, ### P,0.001, NAC, DIC pretreatment compared with TSA treatment alone, NQO1 siRNA pretreatment compared with control siRNA pretreatment, H596-NQO1 cells compared with H596-vector cells; ** P,0.01, *** P,0.001, TSA treatment compared with control cells). doi:10.1371/journal.pone.0042138.g002 induced by TSA. P53 siRNA had no effect on TSA induced disruption of MMP, suggesting a p53-independent mechanism. TSA treatment induced a time and concentration dependent upregulation of pro-apoptotic Bax and downregulation of pro- survival Bcl-xl in an NQO1 and ROS dependent but p53 independent manner (Fig. S1, Fig. 6A). This lead to a dramatic increased ratio of Bax/Bcl-xl, which is well known an important factor in governing cell apoptosis via the disruption of mitochon- dria [36]. Mitochondria disruption then lead to the release of cytochrome c from mitochondria, as evidenced from the cytosolic increase but mitochondrial decrease of cytochrome c (Fig. 6B). Caspase activation was then analyzed by an activity assay using substrates of active cleaved caspase and a western blot analysis of PARP cleavage (Fig. 6A, and Fig. 7A, B). TSA dose dependently increased the activities of caspase-3, -9, and -8. The concentration dependent induction of PARP cleavage further supported caspase-3 activation. The pan-caspase inhibitor, z-VAD-fmk, largely attenu- ated TSA induced apoptosis (Fig. 7C). Altogether, these results suggest that TSA induced apoptotic cell death is caspase-mediated. Consistent with other aspects tested in our study, TSA induced collapse of MMP, cytochrome c release, and caspase activation were all NQO1 and ROS dependent but p53 independent. Antitumor effect of TSA in vivo is NQO1-dependent To confirm the role of NQO1, the antitumor efficacy of TSA was further investigated in A549 tumor xenografts by TSA treatment alone or in combination with DIC (Fig. 8A). TSA (20 mg/kg) treatment could significantly suppress the tumor growth; after 20 days treatment, the volume of tumors in TSA treated group was of 279646 mm3 (P,0.01, compared with control group of 3 552690 mm ). DIC treatment alone also slightly inhibited the 3 tumor growth characterized with the tumor volume of 439699 mm ; to the contrary, DIC pretreatment significantly antagonized the tumor suppression effect of TSA, suggesting that the antitumor effect of TSA in vivo was also NQO1 dependent. Histological examinations showed great tumor necrosis and apoptosis appeared in TSA treated group of mice, and of note, no toxicity in normal organ tissues was observed upon TSA treatment for 20 days (Fig. 8B). PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e42138