TSA as an NQO1 Specific Target Agent Figure 7. TSA caused caspase-dependent apoptosis. A and B, caspase activity test; 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. C, TUNEL assay of TSA induced apoptosis with or without pretreatment of pan-caspase inhibitor z-VAD-fmk. Data are shown as mean 6 SE of at least three independent experiments. (## P,0.01, ### P,0.001, DIC, NAC or z-VAD-fmk pretreatment compared with TSA treatment alone, NQO1 siRNA pretreatment compared with control siRNA pretreated cells; * P,0.05, ** P,0.01, *** P,0.001, TSA treatment compared with blank control cells). doi:10.1371/journal.pone.0042138.g007 problem for TSA development as an effective anti-cancer drug may be of its poor oral bioavailability because of its poor water solubility and extensive intestinal first pass metabolism [31]. Nevertheless, some available pharmaceutical strategies such as TSA nanoemulsions [51] may improve its pharmacokinetic property and thus its druggability. Materials and Methods Ethics statement All animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical University. The use and care of experimental animals complied with all regulations in the Use of Laboratory Animals adopted and promulgated by the United States National Institutes of Health. Cell lines and Culture Human NSCLC cell lines A549 (NQO1+) and NCI-H596 (NQO12) were obtained from the American Type Culture Collection (ATCC, USA). Isogenically matched NQO1-expressing H596 cells were generated by infecting the log-phase H596 cells with lentiviral vectors expressing NQO1 or empty vector. Stable pooled populations Lenti6.3-NQO1-IRES-EGFP (H596-NQO1) or Lenti6.3-IRES-EGFP (H596-vector) were selected in 4.0 mg/mL of blasticidin S. HCL. Cells were grown in RPMI-1640 (Gibco, USA) medium with 10% fetal bovine serum (Hyclone, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (complete media) at 37uC in a humidified atmosphere with 5% CO2. Chemicals and Reagents TSA was from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dicoumarol (DIC), N-acetyl cysteine (NAC), 2, 6-Dichlorophenolindophenol (DCPIP), b-lapachone (Lap), 29,79-Dichlorofluorescein diacetate (DCFH-DA), pifithrin-a (PFT-a), z-VAD-fmk, and Blasticidin. S. HCL were all from Sigma (St. Louis, MO, USA). Antibodies against NQO1 (epitomics, CA, USA), p53 (Millipore, MA, USA), GAPDH and b-actin (Boster Biology, Wuhan, China), Bcl-xl, Bax, PARP, cleaved PAPR, and cytochrome c (Cell Signaling Technology, MA, USA), and COXIV (beyotime, Jiangsu, China) were used. Measurement of NQO1 specific activity NQO1 activity was measured as the rate of DIC-inhibitable DCPIP reduction in cell cytosolic samples as described previously [52]. The reaction was started by the addition of DCPIP, and the reduction of DCPIP was measured at room temperature for 2 min at 600 nm. The DIC-inhibitable part of DCPIP reduction was used to calculate NQO1 activity expressed as nmol DCPIP per mg protein per minute. Cytotoxicity assay Exponentially growing cells were seeded at 8000 cells/well onto a 96-well plate (Costar) and kept overnight. The cells were incubated with TSA or Lap at indicated concentrations. After incubation for 72 h, 20 mL MTT (5 mg/mL) was added to each well and the plate was incubated at 37uC for another 4 h. MTT PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e42138