controlled by caspase activation, Huh7 cells were pre-incubated with pan-caspase inhibitor z-VAD-FMK (50 mM) for 1 h, followed by various stimuli as indicated. DNA ladder assays (Fig. 2A), DNA fragmentation analysis (Fig. 2B and 2C) and FACS analysis (Fig. 2D and 2E) demonstrated that apoptosis induced by combination treatment with Lexa and CHX were totally blocked by pan-caspase inhibitor z-VAD-FMK, suggesting that CHX- sensitized and Lexa-induced apoptosis is caspase dependent. Meanwhile, we measured the effects of caspase 8 specific inhibitor z-IETD-FMK on apoptosis induced by Lexa and CHX combi- nation treatment. As shown in Fig. 2A, 2B, 2C, 2D, and 2E, blockade of caspase 8 activation completely abolished Lexa and CHX combination-induced apoptosis in Huh7 cells. Taken together, these results suggest that caspase activation plays a key role in Lexa-mediated apoptosis induction in HCC cells. Lexa and CHX combination treatment-induced apoptosis is regulated by ROS To assess whether ROS plays a major role in regulating Lexa and CHX combination-induced apoptosis, HCC cells were treated with Lexa and CHX for 6 h. Intracellular ROS levels were determined by a DHED-based fluorescence assay. As shown in Fig. 3A, Lexa and CHX combination treatment induced ROS dramatic increase in apoptotic cells, but not in non-apoptotic cells, suggesting ROS may contribute to CHX-and Lexa-induced CHX and Lexa Combination-Induced Apoptosis in HCC apoptosis. To further determine whether generation of ROS can regulate apoptosis, N-acetyl-L-cysteine (NAC), a precursor of reduced glutathione (GSH) widely used as a thiol-containing antioxidant to scavenge intracellular ROS, was added before administration of Lexa and CHX. As shown in Fig. 3B, the ROS increase induced by combined treatment of Lexa and CHX was completely negated. More importantly, apoptosis assays indicated that the blockade of ROS by NAC can also protect cells against Lexa-and CHX-induced apoptosis (Fig. 3C, 3D, 3E, and 3F). These results suggest that Lexa and CHX combination treatment- induced ROS increase can trigger apoptosis in HCC cells. Effects of combination treatment with Lexa and CHX on protein expression in HCC cells Since CHX is a protein synthesis inhibitor, it may sensitize Lexa to induce apoptosis through down-regulating anti-apoptotic proteins. We analyzed the apoptosis associated protein expression changes in HCC cells treated with Lexa alone or combination- treated with Lexa and CHX for up to 6 h. Western blotting results showed that there were no decreases in anti-apoptotic molecules Bcl-xL or survivin expression; of the pro-apoptotic proteins, both Bad and Bim were down-regulated, while Bak was down-regulated with Lexa single treatment and up-regulated with combination treatment. Additionally, protein levels of pro-apoptotic Bax were not affected, and the expression levels of death receptors DR4 and Figure 2. Lexa and CHX combination treatment induces caspase-dependent apoptosis. A, Huh7 cells were treated with different conditions as indicated. Apoptosis was tested by DNA ladder assays as described in ‘Materials and Methods’. B, Huh7 cells were treated with various stimuli as indicated (Lexa, (1 mg/ml), CHX, (10 mg/ml), or caspase 8 inhibitor IETD (50 mM)). Apoptotic cells were examined as described in Fig. 1B (representative apoptotic cells were marked with arrows). C, Huh7 cells were treated with conditions as indicated and apoptotic ratio was determined by counting cells with apoptotic nuclei as described in ‘Materials and Methods’. Data represent the mean values of three independent experiments (*p,0.05). D, Huh7 cells were treated with various conditions as indicated (Lexa, (1 mg/ml), CHX, (10 mg/ml), IETD (50 mM), z-VAD (50 mM)). Apoptosis was evaluated as in Fig. 1C. E, Huh7 cells were treated with different conditions as indicated and apoptosis percentage was determined as in Fig. 1D. Data represent the mean values of three independent experiments (**p,0.05). doi:10.1371/journal.pone.0016966.g002 PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e16966