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Irreversible inhibition of cytosolic thioredoxin reductase 1 as a mechanistic basis for anticancer therapy

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Science Translational Medicine  14 Feb 2018:
Vol. 10, Issue 428, eaaf7444
DOI: 10.1126/scitranslmed.aaf7444
  • Fig. 1 Correlation of TXNRD1 inhibition with cytotoxicity.

    (A) Structures of inhibitors [TXN reductase 1 (TXNRD1) inhibitor 1 (TRi-1) and TRi-2], inactive analogs (TRi-62 and TRi-79), and a nonspecific analog also inhibiting glutathione disulfide reductase (GSR) (TRi-55). (B to G) Comparative inhibitory activities of TRi compounds and their analogs in enzymatic and cell culture assays. (B) TRi-1 and its analogs tested for inhibition of TXNRD1 or (C) GSR inhibition. (D) TRi-1 with analogs tested for cytotoxicity toward FaDu cells after 72 hours of incubation. (E) TRi-2 and its inactive analog TRi-79 tested for inhibition of TXNRD1 and (F) GSR inhibition. (G) Cytotoxicity toward FaDu cells after 72 hours of incubation with TRi-2 or TRi-79. Results from recombinant enzyme and cell culture experiments are shown as averages of triplicates ± SEM.

  • Fig. 2 Specificity of TXNRD1 targeted inhibition.

    (A to C) TXNRD1 activity in the presence of TXN1 and glutathione (GSH) examined in a TXN1-coupled insulin reduction assay. (A) Ten micromolar TRi-1, TRi-2, or auranofin was preincubated with GSH before incubation with TXNRD1, NADPH, TXN1, and insulin. (B) Incubation of compounds in the presence of TXNRD1, NADPH, and GSH before the addition of TXN1 and insulin. (C) Incubation of compounds in the presence of TXNRD1, NADPH, TXN1, and GSH simultaneously before the addition of insulin. (D and E) Comparisons in the inhibition of human TXNRD1 versus human TXNRD2 using DTNB as a substrate with either TRi-1 (D) or auranofin (E). (F) Compound inhibitory activity toward GPX1. (G) Inhibition of cellular TXNRD activity in FaDu cells after 3 hours of compound exposure. Activity was determined using the insulin end point assay. (H) Cellular GSH concentrations after exposure of FaDu cells to compounds for 6 hours at 10 × IC50. Results from recombinant enzyme and cell culture experiments are shown as averages of triplicates ± SEM. (I to L) Western blot analyses of FaDu cells treated with compounds for 6 hours, examining c-Jun N-terminal kinase (JNK) phosphorylation (I), total JNK (J), p38 phosphorylation (K), and total p38 (L).

  • Fig. 3 SecTRAP formation, cellular H2O2 production, and mitochondrial function.

    (A) TXNRD1 activity with DTNB as a model substrate analyzed before (black bars) and after desalting (white bars) upon incubation with compounds in the presence or absence of NADPH. (B) SecTRAP activity as indicated by juglone reduction measured after desalting upon preincubations of NADPH-reduced TXNRD1 with enzyme inhibitors at concentrations completely inhibiting native TXNRD1 activity. (C and D) H2O2 production in cultured FaDu cells treated with TXNRD1 inhibitors, measured with an Amplex red assay in a concentration-dependent (C) or time-dependent (D) manner. (E and F) Mitochondrial respiration in HCT116 cells using a Seahorse assay, examining adenosine 5′-triphosphate (ATP)–coupled basal respiration and maximal respiratory capacity determined after a 30-min (E) or a 5-hour (F) exposure time. Results are shown as the averages of three experiments performed in triplicate (n = 3), and differences were compared to vehicle using an unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

  • Fig. 4 TRi-1, TRi-2, and auranofin cancer cell cytotoxicity and potency against noncancerous cell lines.

    (A to C) Viability of selected human carcinoma cell lines was determined after 72 hours of incubation with TRi-1 (A), TRi-2 (B), or auranofin (C) to determine dose-dependent cytotoxicity profiles. (D to F) Comparison of cytotoxicities toward TXNRD1-overexpressing human A549 lung carcinoma cells or MEFs with (Txnrd1fl/fl) and without (Txnrd1−/−) the gene encoding for mouse TXNRD1 expression using TRi-1 (D), TRi-2 (E), or auranofin (F). (G to I) Viability of normal human primary fibroblasts, primary keratinocytes, and colon epithelial CCD841 cells after 72 hours of incubation with TRi-1 (G), TRi-2 (H), or auranofin (I). The averages of the cancer cell viability data for the eight cancer cell lines shown in (A) to (F) were pooled and plotted here together with the normal cells for reference.

  • Fig. 5 Rapid anticancer effects of TXNRD1 inhibitors in FaDu xenograft-bearing mice.

    (A) Human FaDu cell xenograft growth in SCID mice, treated twice daily for 4 days starting 13 days after inoculation (“day 0”) with TRi-1 (10 mg/kg) (n = 6), TRi-2 (15 mg/kg) (n = 5), auranofin (10 mg/kg) (n = 5), or vehicle (n = 3). The graph displays percentage increase in tumor growth compared to day 0 (average ± SEM). Groups were compared with a repeated-measures analysis of variance (ANOVA) and Tukey’s multiple comparisons posttest (*P < 0.05, **P < 0.01). (B) Representative PET image of [2-18F]-2-fluoro-2-deoxy-d-glucose ([18F]-FDG) uptake in a FaDu cell xenograft tumor (T), heart (H), and brain (Br), with elimination of radiotracer through kidneys (K) and bladder (Bl). (C) Side-by-side [18F]-FDG uptake in individual tumors on day 0 and day 3, treated with TRi-1 (5 mg/kg) (n = 8) or vehicle (n = 9) once daily. (D) Relative [18F]-FDG uptake in the viable parts of the tumor masses shown in (C) analyzed using a paired t test. (E) Representative three-plane image of a TRi-1–treated low-uptake tumor core with sagittal (S), coronal (C), and transaxial (T) projection. (F) Caspase-3 staining in excised FaDu cell xenografts from mice treated with TRi-1 (5 mg/kg) once daily, intraperitoneally, from day 0 to day 3 (n = 4) or day 4 (n = 4). Five image fields per xenograft were quantitated using ImageJ analysis software and grouped according to treatment duration.

  • Fig. 6 Anticancer effects in PyMT-MMTV syngeneic and MDA-MB-231 xenograft-bearing mice.

    (A to C) Average mammary gland tumor volumes over time in individual PyMT-MMTV mice treated intraperitoneally twice a week with vehicle (n = 7) (A), TRi-1 (5 mg/kg; n = 6) (B), or auranofin (10 mg/kg; n = 8) (C). Mammary tumor volume was measured at a minimum of four nodes per mouse at regular intervals for 20 to 22 days and upon sacrifice. (D) Waterfall plot of averaged tumor volumes from the PyMT-MMTV mice surviving 20 to 22 days. (E) Grouped averaged mammary gland tumor volumes of mice surviving 20 to 22 days compared using an ordinary one-way ANOVA with Tukey’s multiple comparisons posttest. N.S., not significant. (F to H) Tumor volumes of MDA-MB-231 xenografts in athymic mice treated daily for 5 days followed by 2 days of no treatment and then treatment three times per week for 2 weeks with vehicle (F), TRi-1 (10 mg/kg, intravenously) (G), or TRi-1 (10 mg/kg, intraperitoneally) (H). (I) Waterfall plot of the final MDA-MB-231 xenograft tumor volumes after treatment for 22 days. (J) Tumor volumes were compared to vehicle using a two-way ANOVA with Tukey’s multiple comparison posttest (***P < 0.001, ****P < 0.0001, n = 12 in each group). IV, intravenously; IP, intraperitoneally.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/428/eaaf7444/DC1

    Materials and Methods

    Fig. S1. Structures and activities of TRi-1, TRi-2, and analogs.

    Fig. S2. Compound reactivity with reduced GSH.

    Fig. S3. Inhibition of human TXNRD1 and TXNRD2 by TRi-1 or auranofin.

    Fig. S4. Inhibition of cellular TXNRD in HCT116 cells after 3 hours of treatment.

    Fig. S5. TXNRD1 thermostabilization with inhibitors.

    Fig. S6. Comparison of cytotoxicity profiles of TXNRD1 inhibitory small molecules within the NCI-60 cancer cell panel.

    Fig. S7. FaDu cell colony formation assay.

    Fig. S8. GSH depletion in FaDu cells using preincubation with BSO.

    Fig. S9. Average mouse weights during repeated-dose toxicity study.

    Fig. S10. Average mouse weights during MDA-MB-231 xenograft study in athymic mice.

    Table S1. Top 53 compounds from the TXN reductase inhibitor high-throughput screen.

    Table S2. SMILES of TRi-1, TRi-2, and analogs.

    Table S3. Mouse liver microsome stability of TRi-1, TRi-2, and auranofin.

    Table S4. Growth inhibition for TRi-1, TRi-2, and auranofin tested with the NCI-60 cancer cell panel.

    Table S5. Death of mice treated with the combination of auranofin and BSO.

    Table S6. Comparisons of effects between TRi-1 and auranofin.

    References (5762)

  • Supplementary Material for:

    Irreversible inhibition of cytosolic thioredoxin reductase 1 as a mechanistic basis for anticancer therapy

    William C. Stafford, Xiaoxiao Peng, Maria Hägg Olofsson, Xiaonan Zhang, Diane K. Luci, Li Lu, Qing Cheng, Lionel Trésaugues, Thomas S. Dexheimer, Nathan P. Coussens, Martin Augsten, Hanna-Stina Martinsson Ahlzén, Owe Orwar, Arne Östman, Sharon Stone-Elander, David J. Maloney, Ajit Jadhav, Anton Simeonov, Stig Linder, Elias S. J. Arnér*

    *Corresponding author. Email: elias.arner{at}ki.se

    Published 14 February 2018, Sci. Transl. Med. 10, eaaf7444 (2018)
    DOI: 10.1126/scitranslmed.aaf7444

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Structures and activities of TRi-1, TRi-2, and analogs.
    • Fig. S2. Compound reactivity with reduced GSH.
    • Fig. S3. Inhibition of human TXNRD1 and TXNRD2 by TRi-1 or auranofin.
    • Fig. S4. Inhibition of cellular TXNRD in HCT116 cells after 3 hours of treatment.
    • Fig. S5. TXNRD1 thermostabilization with inhibitors.
    • Fig. S6. Comparison of cytotoxicity profiles of TXNRD1 inhibitory small molecules within the NCI-60 cancer cell panel.
    • Fig. S7. FaDu cell colony formation assay.
    • Fig. S8. GSH depletion in FaDu cells using preincubation with BSO.
    • Fig. S9. Average mouse weights during repeated-dose toxicity study.
    • Fig. S10. Average mouse weights during MDA-MB-231 xenograft study in athymic mice.
    • Table S1. Top 53 compounds from the TXN reductase inhibitor high-throughput screen.
    • Table S2. SMILES of TRi-1, TRi-2, and analogs.
    • Table S3. Mouse liver microsome stability of TRi-1, TRi-2, and auranofin.
    • Table S4. Growth inhibition for TRi-1, TRi-2, and auranofin tested with the NCI-60 cancer cell panel.
    • Table S5. Death of mice treated with the combination of auranofin and BSO.
    • Table S6. Comparisons of effects between TRi-1 and auranofin.
    • References (5762)

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