Vorinostat Induces DNA Damage in AML Cells Figure 4. Vorinostat mediated DNA damage and apoptosis is in part due to reactive oxygen species. A NB4 and U937 cells were treated with vorinostat for 6 h and 18 h and stained with DCF-DA to quantify intracellular peroxides. B NB4 and U937 cells were treated with vorinostat for 6 h, 12 h and 18 h and induction of HO-1 was assessed by western blot. b-Actin was used as a loading control. C U937 cells were pre-treated with 2 mM N-acetyl cysteine (NAC), an antioxidant, followed by 2.0 mM vorinostat for 18 h. Induction of cH2AX was assessed by western blot. b-Actin was used as a loading control. D Cells were stained with PI after 24 h of exposure to vorinostat and/or N-acetyl cysteine, and apoptosis was quantified as percentage of cells with Sub-Go DNA content. Asterisks show significant difference (** ,0.01 *** ,0.001). doi:10.1371/journal.pone.0020987.g004 Vorinostat induces reactive oxygen species and antioxidants reduce vorinostat-induced DNA damage and apoptosis Previous studies have reported that HDACi treatment results in ROS production and ROS has subsequently been postulated to be a key mechanism in HDACi-induced apoptosis [3]. We analyzed the production of peroxides (mainly hydrogen peroxide) by DCF- DA staining in NB4 and U937 cells after treatment with vorinostat for 6 h or 18 h. Consistent with the induction of the oxidative stress DNA adduct 8-oxo-G (Figure 1D), we observe an increase in peroxides beginning at 6 h in both cell lines (Figure 4A). To validate this finding we assayed for HO-1 protein expression, a marker of cellular oxidative stress. Treatment of NB4 and U937 cells with vorinostat resulted in the up-regulation of HO-1 protein beginning at 6 h (Figure 4B). Augmented ROS levels can be due to a decrease in intracellular GSH, however, at a time point preceding the appearance of ROS, vorinostat was not found to alter GSH levels (Figure S1A). If left unrepaired, DNA adducts including 8-oxo-G can give rise to DSBs, the most cytotoxic DNA lesion. To determine if ROS- induced DNA damage may contribute to vorinostat-induced apoptosis, NB4 cells were pre-treated with the antioxidant NAC for 1 h prior to treatment with vorinostat. Pre-treatment with this antioxidant resulted in a significant reduction in vorinostat- induced DSBs, as measured by cH2AX protein expression (Figure 4C). Pre-treatment with NAC was also associated with a reduction of cells arresting at the G2-M cell cycle phase (Figure S1B) and a partial protection in vorinostat-induced apoptosis (Figure 4D). In addition to NAC, the antioxidants L-GSH and MnTMPyP, a superoxide dismutase mimetic, also protected against vorinostat induced apoptosis in NB4 and U937 cells (Figure S1C). PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20987