TSA as an NQO1 Specific Target Agent Figure 5. TSA activates an NQO1-initiated, ROS-mediated, and p53- independent mitochondrial membrane potential disruption (JC-1 assay). 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. Cells treated as indicated for 24 h were loaded with 3 mM of JC-1 for 15 min at 37uC, then softly washed with PBS for two times. In the cytosol, the monomeric form of this dye fluoresces green and within the mitochondrial matrix, highly concentrated JC-1 forms aggregates that fluoresce red. A, representative photos taken by Leica BMI3000 B microscope with a confocal microscope application. B, the fluorescence was read at 488 nm excitation and 530 nm emission for green, and at 540 nm excitation and 590 nm emission for red using a Synergy-H1 fluorimeter (Bio-Tek Instruments) and the changes in mitochondrial potential were calculated as the red/green ratio for each condition. (## P,0.01, ### P,0.001, DIC, NAC pretreatment compared with TSA treatment alone or NQO1 siRNA pretreatment compared with control siRNA pretreated cells; ** P,0.01, *** P,0.001, TSA treatment compared with control cells). doi:10.1371/journal.pone.0042138.g005 down-regulating p53 protein levels warrant further research, the present results indicate that this effect may be caused by NQO1 regulated ubiquitin-independent p53 degradations, because TSA treatment also leaded to the down-regulation of NQO1 in parallel to the change of p53 protein levels (Fig. S3). NQO1 could stabilize p53 by preventing a ubiquitin-independent proteasomal degrada- tion [15,44]. Consistently, the NQO1 inhibitor like DIC could promote this degradation pathway and thus interfere with p53 initiated apoptotic pathway [45]. Our findings that TSA synergizing with DIC on reducing p53 protein levels provides reasonable explanation why NQO1 substrates induced apoptotic cell death is p53 independent. It may suggest that NQO1 substrates, like typical NQO1 inhibitors, can also promote the ubiquitin-independent proteasomal degradation; detailed mecha- nisms are currently under investigation in our laboratory. Since caspase serves as a central component of apoptosis machinery, we next sought to determine its role on TSA induced apoptotic cell death. Unlike the NQO1 substrate Lap which induced caspase independent apoptosis [46], TSA led to a concentration dependent activation of caspase-3, -8, and -9. In addition, pretreatment of the pan-caspase inhibitor z-VAD-fmk largely prevented TSA induced apoptosis that further demon- strated a caspase-dependent apoptosis upon TSA treatment (Fig. 7). Consistent with the results of apoptosis assays, the activation of caspases was also found also NQO1 and ROS dependent but p53 independent. Mitochondria mediated apopto- sis is one crucial apoptotic mechanism involving activation of caspases. A previous study Chiu et al. demonstrated that TSA induced apoptosis of A549 cells was characterized with ROS production, DNA damage, and MMP loss [35], indicating that the ROS triggered mitochondrial pathway may play an important role on TSA induced cell death. For this consideration, we have made detailed studies herein to determine the role of NQO1 and p53 on initiating and programming the mitochondrial apoptotic cell death pathway. Our study showed that TSA treatment not only led to an NQO1 dependent and p53 independent loss of MMP and the subsequent release of cytochrome c from mitochondria (Fig. 5 and Fig. 6B), but also cause a p53 independent increased ratio of pro- apoptotic protein Bax to pro-survival protein Bcl-xl (Fig. 6A), which were considered to initiate apoptotic cell death [36]. These results in combination strongly suggest that the mitochondrial apoptotic pathway plays an important role on mediating TSA induced NQO1- and caspase-dependent but p53 independent apoptotic cell death in NSCLC cells. Although the detailed molecular mechanisms underlying TSA induced apoptosis warrant further research to explore, our results clearly indicate that some p53 backup systems can be activated by NQO1 triggered redox cycles and involving factors in the conditions where p53 is largely impaired due to NQO1 substrates induced instabilization. Taking together, it can be proposed that TSA is able to induce a p53 PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e42138