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 (IC50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC50 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.

Keywords: NRF2; colorectal carcinoma; glutathione; pentose phosphate pathway; thioredoxin.

Figure 1

Five classes of glutathione S‐transferase are up‐regulated in colorectal cancer tissue‐originated spheroids (CTOSs), whereas three classes are down‐regulated. mRNA expression of each gene was quantified by real‐time RT‐PCR and expression level was indicated as relative Log₂ expression. Black and gray box plots represent fibroblasts (embryonic or dermal) and CTOSs (from distinct colorectal carcinomas patients), respectively. (A) GST isoforms up‐regulated in colorectal CTOSs compared to fibroblasts. (B) GST isoforms down‐regulated in colorectal CTOSs compared to fibroblasts. GST, glutathione S‐transferase; DF, TIG111, and 119, dermal fibroblasts; TIG3 and OUMS36, embryonic fibroblasts.

Figure 2

Pharmacological inhibition of redox regulation up‐regulated NRF2 target genes (glutathione system) in colorectal cancer tissue‐originated spheroids (CTOSs). Colorectal CTOSs were incubated with designated concentration of inhibitor. mRNA expression of each gene was quantified by real‐time RT‐PCR and expression level was indicated as relative Log₂ expression. Each plot indicates Log₂ expression of each CTOS (i.e., data from distinct patients). (A and B) Expression of GCLC. (C and D) Expression of GCLM. (A and C) Drug exposure for 24 h. (B and D) Drug exposure for 48 h. Asterisk denotes significant difference from untreated group. Aura: auranofin (TXNRD inhibitor), CB: CB83 (G6PD inhibitor), Pipe: piperlongumine (GST π inhibitor).

Figure 3

Pharmacological inhibition of redox regulation up‐regulated NRF2 target genes (thioredoxin system) in colorectal cancer tissue‐originated spheroids (CTOSs). Colorectal CTOSs were incubated with designated concentration of inhibitor. mRNA expression of each gene was quantified by real‐time RT‐PCR and expression level was indicated as relative Log₂ expression. Each plot indicates Log₂ expression of each CTOS (i.e., data from distinct patients). (A and B) Expression of PRDX1. (C and D) Expression of TXN. (E and F): expression of TXNRD1. (A, C, and E) Drug exposure for 24 h. (B, D, and F) Drug exposure for 48 h. Asterisk denotes significant difference from untreated group.

Figure 4

Pharmacological inhibition of redox regulation up‐regulated NRF2 target genes (NADPH production) in colorectal cancer tissue‐originated spheroids (CTOSs). Colorectal CTOSs were incubated with designated concentration of inhibitor. mRNA expression of each gene was quantified by real‐time RT‐PCR and expression level was indicated as relative Log₂ expression. Each plot indicates Log₂ expression of each CTOS (i.e., data from distinct patients). (A and B) Expression of G6PD. (C and D) Expression of IDH1. (E and F) Expression of ME1. (A, C, and E) Drug exposure for 24 h. (B, D, and F) Drug exposure for 48 h. Asterisk denotes significant difference from untreated group.

Figure 5

Dual inhibition of GST π and TXNRD results in synergistic cell death in colorectal cancer tissue‐originated spheroids (CTOSs). Cells were treated with auranofin and piperlongumine. Box plot indicates Combination Indices (CI) with molar ratios (auranofin:piperlongumine) from 1:20 to 2:5. Black and gray boxes represent fibroblasts (embryonic or dermal) and CTOSs (from distinct CRC patients), respectively. A perpendicular gray pillar is located around CI from 0.9 to 1.1, where dual inhibition displays “nearly additive” effect. CIs to the left of the pillar (<0.9) signify “synergism”. The lower the CI, the stronger the synergism. Asterisk denotes significant difference between CTOSs and fibroblasts.

Figure 6

Selective killing of colorectal cancer tissue‐originated spheroids (CTOS) cells by dual inhibition of GST π and TXNRD. Colorectal CTOSs or fibroblasts were incubated with designated concentration of each inhibitor (molar ratio: A: 1:5, B: 1:10, and C: 1:20). Scatter plots indicate calculated percentage of survived cells (drugs treated/untreated). Experiments were repeated with different passage points. Note that colorectal CTOSs are more “sensitive” to drugs' treatment than fibroblasts are, indicating selective killing of cancer cells. Error bar represents mean ± standard error of the mean. OUMS36 and TIG3: embryonic fibroblasts, TIG119, 111, 102 and DF, dermal fibroblasts. Asterisk denotes significant difference between CTOSs and embryonic fibroblasts. TXNRD, thioredoxin reductase.

Figure 7

Prolonged incubation of GST π/TXNRD inhibitors induces drug resistance and double‐strand DNA breaks in colorectal cancer tissue‐originated spheroids (CTOS) cells. Colorectal CTOS cells were incubated with designated molar ratio and serial increment of two inhibitors for 7 days. Although majority of CTOS cells were killed during prolonged exposure to drugs, small number of cells display drug resistance. Whole‐cell proteins were extracted from such “persisters” and each well was loaded with same amount of proteins (20 μg for H2A.X, 30 μg for H2A, and 10 μg for H3). Phosphorylation of histone H2 (H2A.X, a marker of double‐strand DNA breaks) was visualized by western blotting. Blotting for histone H2A and H3 serve as loading controls. TXNRD, thioredoxin reductase.

Figure 8

Dual inhibition of GST π and TXNRD decreased CD44v9 surface presentation of colorectal cancer tissue‐originated spheroids (CTOSs). Colorectal CTOSs were incubated with designated concentration of each inhibitor (molar ratio 1:20 or 1:10). Enzymatically dispersed cells were stained CD44v9 antibody and the cell surface fluorescent signal was analyzed with flow cytometry. (A) CD44v9‐positive rate (see Fig. S4 for gating strategy). (B) Mean fluorescent intensity of CD44v9^high population (Fig. S4). Asterisk denotes significant difference between untreated and dual inhibition (paired t).

Figure 9

Dual inhibition of GST π and TXNRD delayed tumor formation and growth in vivo. Colorectal cancer tissue‐originated spheroids (CTOSs) were incubated with auranofin 0.5 μmol/L + piperlongumine 5 μmol/L for 7 days. (A) Drug‐resistant CTOSs retained spheroid structure (AP) similar to untreated (ϕ) CTOSs. (B) Pairwise comparison of tumor formation between untreated (ϕ) and dual inhibition (AP). (C) Collected tumors. (D) Tumor weights after collection (P = 0.008 by paired t test) Scale bar: 100 μm. TXNRD, thioredoxin reductase.