Figure 1.
TSA induced cytotoxicity is NQO1 dependent, ROS mediated, and p53-independent (MTT assay).
A, protein levels and enzyme activities of NQO1 in NSCLC cells. NQO1 activity <1.0 nmol⋅min−1⋅µg−1 was indicated non-detectable (ND). B, cytotoxicity of TSA and Lap in NSCLC cells; cells were exposed to gradient concentrations of TSA (0.4–40 µM) or Lap (0.5–10 µM) for 72 h. C, effects of NAC or DIC pretreatment; A549 cells were pretreated with 5 mM of NAC for 1 h or 10 µM of DIC for 30 min. D, effects of NQO1 silence in A549 cells. E, TSA cytotoxicity in H596 and NQO1-transfected H596 cells. F, effect of P53 inhibition or silence; A549 cells were pretreated with 10 µM of PFT-α for 5 h or pretreated with P53 siRNA. Data are shown as mean ± SE of three independent experiments.
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 µM of DIC for 30 min, 5 mM of NAC for 1 h, or 10 µM of PFT-α for 5 h and then exposed to 40 µM 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 ± 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).
Figure 3.
TSA induces NQO1-dependent and ROS mediated DNA damage.
DNA lesions determined by alkaline comet assays. A, TSA induced DNA damage in A549 and H596 cells, as well as the effect of NAC or DIC pretreatment. B, effect of NQO1 silence in TSA induced DNA damage in A549 cells. C, DNA damage in H596-NQO1 and H596-vector cells. Left panel, the representative morphological appearance of cells with TSA treatment for 24 h; right panel, quantitative results expressed as fold change (T/C) of comet tail lengths or expressed as comet tail lengths (arbitrary unit). Data are shown as mean ± SE of at least three independent experiments. (# P<0.05, ## P<0.01 NQO1 silence vs. control siRNA or H596-NQO1 vs. H596-vector; * P<0.05, ** P<0.01, *** P<0.001, TSA or H2O2 treatment compared with control cells).
Figure 4.
TSA induces NQO1 dependent generation of ROS in NSCLC cells.
Intracellular ROS generation measured by DCF staining. A, for time course study, cells were treated with 10 µM of TSA with or without pretreatment by 10 µM of DIC or 5 mM of NAC; B, cells were exposed to indicated doses of TSA for 1 h; C, effect of NQO1 silence on TSA triggered ROS formation; cells were exposed to 4 µM or 40 µM of TSA for 1 h; D, H595-NQO1 cells and H596-vector cells were exposed to 10 µM or 40 µM of TSA for 1 h. H2O2 (400 µM) was used as a positive control. E and F, Glutathione cycling assays; E, NSCLC cells were exposed to indicated doses of TSA with or without DIC pretreatment for 24 h. F, H596-vector and H596-NQO1 cells were exposed to TSA for 24 h. Data are shown as relative changes to blank controls and expressed as mean ± SE of at least three independent experiments. (# P<0.05, ## P<0.01, ### P<0.001 NQO1 siRNA vs. control siRNA, H596-NQO1 vs. H596-vector, A549 with DIC vs. TSA alone; * P<0.05, ** P<0.01, *** P<0.001, TSA or H2O2 treatment vs. their respective control cells).
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 µM) alone or pretreated with DIC, NAC, or subjected to NQO1 and p53 silence, prior to the treatment of 40 µM of TSA. Cells treated as indicated for 24 h were loaded with 3 µM of JC-1 for 15 min at 37°C, 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).
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 µM) alone or pretreated with DIC, NAC, or subjected to NQO1 and p53 silence, prior to the treatment of 40 µM 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).
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 µM) alone or pretreated with DIC, NAC, or subjected to NQO1 and p53 silence, prior to the treatment of 40 µM 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 ± 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).
Figure 8.
TSA retards the tumor growth NQO1 dependently in A549 tumor xenografted nude mice.
A, upper, tumor volume measurement (P<0.01, TSA vs. control; P<0.05, TSA vs. TSA+DIC); lower, tumor weight (# P<0.05, TSA vs. TSA+DIC, *** P<0.001, TSA vs. control); data are shown as mean ± SE of six mice. B, histological examinations; tumor and tissue sections were analyzed by hematoxylin and eosin staining for histological features. C, biodistribution of TSA in A549 tumor loaded nude mice; data are shown as mean ± SE of 6 mice.
Figure 9.
Proposed mechanisms for TSA-induced apoptotic cell death in NQO1-positive cells.
TSA induces apoptotic cell death of NSCLC cells via a unique NQO1-initiated and ROS-mediated activation of a p53 independent but caspase dependent mitochondrial apoptotic pathway.