Figure 8

Inhibition of extracellular signal-regulated kinase (ERK)1/2 abolishes chemotherapy-induced apoptosis in manganese superoxide disumutase (MnSOD) small-interfering RNA (siRNA)-transfected SKOV-3 cells. (A) Cells transfected with non-specific control (Ctrl siRNA) or MnSOD siRNA in the presence or absence of doxorubicin (DOX, 5 μ M) or paclitaxel (PTX, 0.1 μ M) were analysed for activities of ERK1/2 and Akt by western blotting using antibodies specific for phosphorylated, activated forms of ERK1/2 and Akt. The membrane was then re-probed with total ERK1/2, Akt, and β-actin. Small-interfering RNA-mediated depletion of MnSOD was confirmed by western blot analysis using antibodies specific against MnSOD. (B) Cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with the ERK1/2 inhibitor (PD98059, 50 μ M) for 30 min followed by DOX or PTX addition for another 48 h. Cells were then harvested for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. (C) Cells were transiently transfected with the control vector (pcDNA3.1) or DN-MEK1 for 24 h, and then treated with DOX or PTX for another 48 h. Whole-cell lysates were analysed for levels of phosphorylated and total forms of ERK1/2 by western blotting. β-Actin serves as a protein-loading control. Right, apoptosis was assessed by the TUNEL assay. Experiments were repeated three times, and data are shown as mean±s.d. ^*P<0.05 vs untreated MnSOD siRNA-transfected cells as indicated.