fig4

Figure 4. DCA and THP activates the ROS-JNK signaling pathway. A: Hep3B and Huh7 cells were treated with a combination of DCA and THP or DCA + THP + NAC (10 mmol/L) for 24 h and the protein levels of p-JNK, t-JNK, p-AKT, t-AKT, p-ERK and t-ERK were determined by Western blotting; B-D: quantification of the protein levels of p-JNK (B), p-ERK (C) and p-AKT (D). The data were normalized by t-JNK, t-ERK and t-AKT, respectively. The data were expressed as the fold change against the control group and were represented as the mean ± SEM *P < 0.05; **P < 0.01; E: Hep3B and Huh7 cells were treated with DCA (20 mmol/L), THP (300 nmol/L) or a combination of DCA and THP for 24 h, and the protein levels of p-JNK and t-JNK were determined by Western blotting; F-H: Hep3B and Huh7 cells were treated with a combination of DCA and THP or DCA + THP + NAC or DCA + THP + SP600125 (10 µmol/L) for 24 h, where: protein levels of p-JNK and t-JNK were determined by Western blotting (F); cell viability was determined using the CCK-8 assay (G); and apoptosis was calculated by flow cytometry analysis (H); I: schematic illustration for the synergistic effect of DCA and THP in human liver cancer cells. DCA and THP significantly enhanced ROS levels giving rise to JNK activation and apoptosis induction. This synergistic effect of DCA and THP could be attenuated by co-treatment with antioxidant NAC or JNK kinase inhibitor SP600125. Experiments were repeated at least three times. ^**P < 0.01. DCA: dichloroacetate; THP: pirarubicin; t: total; p: phosphorylated; ROS: reactive oxygen species