Combination of vorinostat and PEITC induced ROS-mediated apoptosis. (A) Illustration of biochemical strategy to enhance cellular ROS accumulation. Vorinostat induced NOX-derived O₂⁻, which is further converted to H₂O₂. PEITC was used to inhibit H₂O₂ elimination by GSH system and thus caused further ROS stress. (B) Treatment with 2μM vorinostat and 2.5μM PEITC for 18 hours caused significant increase of O₂⁻ and H₂O₂ in U937 cells, respectively, as detected by fluorescent probes Het and DCF-DA. (C) Quantitative analysis of H₂O₂ accumulation induced by 2μM vorinostat alone (Vor), 2.5μM PEITC alone, and vorinostat plus PEITC (V + P). Bars represent mean ± SD from 3 experiments. (D) Change of mitochondrial transmembrane potential in U937 cells treated with the indicated compounds was detected by flow cytometry using rhodamine-123. Numbers indicate percentage of cells with loss of transmembrane potential. Pretreatment with 5mM NAC 1 hour before coadministration of vorinostat and PEITC completely blocked the loss of transmembrane potential induced by such combination. (E) Apoptosis induced by the indicated compounds and protection of cell death by pretreatment with 5mM NAC 1 hour before was detected by double staining of annexin V/PI. Numbers indicate percentage of cell death population. (F-G) Incubation with 2μM vorinostat for 18 hours followed by 5μM PEITC for another 2 hours (V18h/P2h) also induced significant increase of H₂O₂ in U937 cells and subsequent decrease of mitochondrial transmembrane potential (V18h/P3h, vorinostat for 18 hours followed by PEITC for 3 hours). Pretreatment with 5mM NAC 1 hour before inhibited both increase of H₂O₂ and loss of mitochondrial transmembrane potential (+NAC). (H) Incubation with 2μM vorinostat for 18 hours followed by 5μM PEITC for 6 hours resulted in tremendous apoptosis as demonstrated by annexin V/PI assay. Pretreatment with 5mM NAC significantly prevented the cell death induced by the combination treatment. Numbers indicate percentage of apoptotic cell death population.