Fig 2: HG reduces mitochondrial thioredoxin reductase 2 (TRXR2) in rMC1. HG exposure of rMC1 for 3 days reduces TRXR2 level compared to LG, while that of TXNIP is increased (first two panels). On the other hand, treatment of rMC1 with triple drug combination TMM increases TRXR2 and decreases TXNIP level under both HG and LG, suggesting a beneficial effect of these drugs on HG-induced redox stress. A representative of n = 3 is shown from fixed-cell confocal imaging at 630× magnification in a Zeiss confocal microscope.

Fig 3: Expression of antioxidant proteins upon cyst(e)inase treatment and their concurrent inhibition. a Comparison of proteins related to antioxidant function in the three PDAC cell lines. Nrf2 nuclear factor (erythroid-derived 2)-like 2, GCL-C glutamate-cysteine ligase catalytic subunit, xCT cystine/glutamate antiporter, TXNRD1 and TXNRD2, thioredoxin reductase 1 and 2, respectively. b, c Expression of antioxidant proteins following cyst(e)inase treatment. TBHP treatment (200 μM, 2 h) is included as a positive control for ROS accumulation b. d−f Isobologram of the effect of the combination of cyst(e)inase (Cys) and sulfasalazine d, cyst(e)inase and BSO e, and cyst(e)inase and auranofin f (data from more than three independent experiments). g Relative mitochondrial ROS (mROS) levels (red) and cell death (black) in MIA-PaCa2 cells 24 h after treatment with 50 nM Cys, 0.5 μM (mROS) or 2 μM (cell death) auranofin (Aur), their combination (Cys + Aur), and the combination plus 0.5 mM GSH ethyl ester (GSH) (for mROS, n = 5 independent experiments; for cell death, n = 3 independent experiments). h Relative mitochondrial ROS (mROS) levels (red) and cell death (black) in BxPC3 cells 24 h after treatment with 100 nM Cys, 2 μM (mROS) or 4 μM (cell death) Aur, their combination (Cys + Aur), and the combination plus 0.5 mM GSH ethyl ester (GSH) (for mROS, n = 5 independent experiments; for cell death, n = 3 independent experiments). i Regulatory cell cycle proteins 24 h after the indicated combinatorial treatments in MIA-PaCa2 and BxPC3 cells. For (b, c), “+” represents 250 nM cyst(e)inase treatment except for (c), where “+” for Panc1–48 h represents 100 nM treatment. ####P or ****P < 0.0001; for (g, h), compared to untreated controls (*, comparison done for Cys, Aur and Cys + Aur) or to Cys + Aur (#, comparison done for only Cys + Aur + GSH); one-way ANOVA with Bonferroni’s method for multiple-comparison test. Experiments in (a, b, c, i) were performed twice. All other experiments were repeated three times or more