PMC

Accumulation of oxidized Ero1α by imexon treated MiaPaCa-2 cells. MiaPaCa-2 cells were incubated with the indicated concentration of imexon for 6h, and separated on a non-reducing acrylamide gel. Diamide treatment was used to identify fully oxidized (OX2) Ero1α (lane 7), while DTT was used to identify fully reduced (OX1) Ero1α (lane 8).

Effect of imexon on ER oxidative response proteins. Pancreatic cell lines were incubated with the indicated concentration of imexon for 24h, and cell lysates were separated by SDS-PAGE and analyzed by immunoblotting. PDI, Ero1α, BiP, and β-actin are 57kDa, 60 kDa, 78 kDa, and 42 kDa, respectively.

Imexon activates an ER stress response. (A) Panc-1, (B) BxPC3, and (C) MiaPaCa-2 were transfected with an ERSE reporter, a negative control, or a positive control for 24h, at which time the cells were washed and then exposed to drugs for an additional 8h with increasing concentrations of imexon. Data shown are the mean ± SEM, n=9 (for MiaPaCa-2 and Panc-1) and n=6 (for BxPC3). *p < 0.05, **p < 0.01.

Effect of imexon on proteins associated with ER translational control. The pancreatic cell lines MiaPaCa-2, Panc-1, or BxPC3 were incubated with the indicated concentration of imexon for 24h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting. Data shown are representative of 3 independent experiments. eiF2B5, eiF2B2, p-EIF2α, eIF2Bα, and β-actin are 85 kDa, 39 kDa, 38 KDa, 38kDa, and 42 kDa, respectively.

Growth inhibitory effects of imexon in pancreatic cell lines with eIF2B5 silencing. Pancreatic cells were transfected with siRNA to eIF2B5 or a non-targeting sequence (si CT) followed by 72h incubation with imexon (IMX). A) Growth inhibition was analyzed by MTT dye metabolism. Data are presented as % of control, where 100% is si CT with no imexon (Mean ± SD, n=8).
Effect of IMX in si CT: *p < 0.05; **p < 0.01;
Effect of IMX in si elF2B5: ^p < 0.05, ^^p < 0.01;
The effect of si elF2B5 vs. si CT is significant at all concentrations of IMX (p < 0.01).
B) Growth inhibition was analyzed by colony-forming assay. Data are presented as % of control, where control cells are mock transfected with transfection reagent but no siRNA (Mean ± SEM, n=3). Effect of IMX: *p < 0.05
C) Western blot analysis of eIF2B5 protein expression in pancreatic cell lines following 24h transfection with siRNA. eIF2B5 and β-actin are 85 kDa and 42kDa, respectively.

Fig. 5A Effects of imexon and eIF2B5 silencing on protein expression. MiaPaCa-2 cells were transfected with eIF2B5 siRNA or a non-targeting sequence (si CT) and examined for protein synthesis. Data are expressed as the percent of control, where the control is untreated cells transfected with a non-targeting siRNA (Mean ± SD, n=8). Imexon significantly inhibited protein synthesis in both si CT and si eIF2B5 transfected cells (*p ≤ 0.05). Knockdown of eiF2B5 also inhibits protein synthesis and is augmented by 250 μM IMX (^p = 0.0017). There is no significant augmentation of synthesis inhibition by 500 μM IMX (p = 0.75, si elF2B5 vs. si CT).
Fig. 5B Effect of NAC supplementation on protein synthesis inhibition by imexon. MiaPaCa-2 cells were pre-treated with 10 mM NAC overnight. NAC was either maintained (“continuous”) or the NAC removed (“pre-treated only”), in the presence of varying concentrations of imexon for 24h. Alternatively, the addition of NAC was delayed until 2h after imexon exposure (“post imexon”). Protein synthesis was measured by ³H-leucine incorporation. Data are expressed as percent of control, where the control is untreated cells (Mean ± SEM, n = 20). *p<0.05, **p<0.01
Fig 5C. Effect of ROS scavengers on protein synthesis inhibition by imexon. MiaPaCa-2 cells were pre-treated for 2h with the 100 U/ml of pegylated superoxide dismutase (PEG-SOD), 200 U/ml of pegylated catalase (PEG-CAT), or 100μM of 1-oxyl-2,2,6,6-tetramethyl-4-hydroxy- piperidine (tempol) before the addition of imexon for a total of 24h. Protein synthesis was measured by ³H-leucine incorporation. Data are expressed as percent of control, where the control is untreated cells (Mean ± SEM, n = 18).