Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells

Abstract

NRF2 stabilizes redox potential through genes for glutathione and thioredoxin antioxidant systems. Whether blockade of glutathione and thioredoxin is useful in eliminating cancer stem cells remain unknown. We used xenografts derived from colorectal carcinoma patients to investigate the pharmacological inhibition of glutathione and thioredoxin systems. Higher expression of five glutathione S‐transferase isoforms (GSTA1, A2, M4, O2, and P1) was observed in xenograft‐derived spheroids than in fibroblasts. Piperlongumine (2.5–10 μmol/L) and auranofin (0.25–4 μmol/L) were used to inhibit glutathione S‐transferase π and thioredoxin reductase, respectively. Piperlongumine or auranofin alone up‐regulated the expression of NRF2 target genes, but not TP53 targets. While piperlongumine showed modest cancer‐specific cell killing (IC 50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC 50 difference between cancer spheroids and fibroblasts: P = 0.002). The synergism of dual inhibition was evaluated by determining the Combination Index, based on the number of surviving cells with combination treatments. Molar ratios indicated synergism in cancer spheroids, but not in fibroblasts: (auranofin:piperlongumine) = 2:5, 1:5, 1:10, and 1:20. Cancer‐specific cell killing was achieved at the following drug concentrations (auranofin:piperlongumine): 0.25:2.5 μmol/L, 0.5:2.5 μmol/L, or 0.25:5 μmol/L. The dual inhibition successfully decreased CD44v9 surface presentation and delayed tumor emergence in nude mouse. However, a small subpopulation persistently survived and accumulated phosphorylated histone H2A. Such “persisters” still retained lesser but significant tumorigenicity. Thus, dual inhibition of glutathione S‐transferase π and thioredoxin reductase could be a feasible option for decreasing the tumor mass and CD44v9‐positive fraction by disrupting redox regulation.

Metabolism-Based Therapeutic Strategies Targeting Cancer Stem Cells

“…Besides glutathione, strategies aimed at inhibiting cellular antioxidants are currently applied with relative success, mostly improving response to conventional therapies (Figure 2). For example, treatment with auranofin, a thioredoxin reductase inhibitor, increased the sensitivity of human breast CSCs to radiotherapy (Rodman et al, 2016), while a synergistic reduction of the CD44v9 + cells content was achieved by inhibiting glutathione-S-transferase and thioredoxin reductase in patient-derived xenograft (PDX) models of CRC (Tanaka et al, 2016). Arsenic trioxide (ATO), an FDA-approved drug for acute promyelocytic leukemia that increases ROS content and depletes superoxide dismutase (SOD) and glutathione peroxidase (GPX) (Li et al, 2006), proved to reduce CSCs content in different cancer types (Ding et al, 2014; Li et al, 2015a; Chang et al, 2016; Bell et al, 2018).…”

The Metabolic Heterogeneity and Flexibility of Cancer Stem Cells

“…The selective Nrf2 inhibitor trigonelline, the xCT inhibitor sulfasalazine, the GSH inhibitor buthionine sulfoximine, and the Trx inhibitor auranofin induce cell death by accumulating ROS in CSCs. When the GSH pathway is inhibited, Trx expression is increased to compensate for it, indicating that the inhibition of both GSH and Trx at the same time can eliminate CSCs more efficiently than the inhibition of GSH or Trx alone [ 182 , 183 , 184 , 185 ].…”

New Challenges to Study Heterogeneity in Cancer Redox Metabolism

“…Multiple other studies have shown this efficacy in combination with common cancer drugs or radiotherapy for multiple cancers (e.g., Lu et al, 2007; Sobhakumari et al, 2012; Rodman et al, 2016; Roh et al, 2016; Tanaka et al, 2016). Together, the intracellular redox states expressed in terms of reducing equivalents and thiol compounds (Box 1) not only influence several signaling processes but also control the reactivities of ROS and their redox partners by regulating ROS homeostasis.…”