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. 2024 Jun 12:14:1394653.
doi: 10.3389/fonc.2024.1394653. eCollection 2024.

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells

Joseph A Haight  1 , Stacia L Koppenhafer  2 , Elizabeth L Geary  2 , David J Gordon  2
Affiliations

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells

Joseph A Haight et al. Front Oncol. .

Abstract

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

Keywords: 4E-BP1; RRM2; auranofin; plk1; protein synthesis; reactive oxygen species.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Hydrogen peroxide inhibits protein synthesis in sarcoma cell lines. (A) EW8 and TC71 cells were treated with hydrogen peroxide, NAC, or the combination for three hours. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (B, C) EW8 (B) and TC71 (C) cells were treated with hydrogen peroxide (100 μM), NAC (5 mM), or the combination for three hours and then labeled with O-propargyl-puromycin for quantification of protein synthesis by flow cytometry. (D) Additional sarcoma cell lines were treated with hydrogen peroxide, NAC, or the combination for three hours and then labeled with puromycin for immunoblotting. (E) EW8 and TC71 cells were treated with hydrogen peroxide for one hour and then protein synthesis was assessed using puromycin. (F) Ewing sarcoma cells were treated with hydrogen peroxide (100 μM) for three hours and then allowed to recover for twenty-one hours before protein synthesis was assessed using puromycin.
Figure 2
Figure 2
Auranofin inhibits protein synthesis in Ewing sarcoma cell lines. (A) Dependency Map data (https://depmap.org/portal/) showing the effect of CRISPR knockout of thioredoxin reductase on the growth of cancer cell lines. (B) EW8 and TC71 cells were treated with auranofin (5 μM) for twenty-four hours, or hydrogen peroxide (100 μM) for three hours, and then ROS was quantified using a DCFDA fluorescence assay and normalized to cells treated with DMSO. Error bars represent the mean ± SD of three independent experiments with six technical replicates per drug treatment. (C) Dose response curves for Ewing sarcoma cells treated with auranofin. Cell viability was assessed 72 hours after drug was added using the AlamarBlue assay. Error bars represent the mean ± SD of three technical replicates. The results are representative of two independent experiments. (D) EW8 and TC71 cells were treated with auranofin, NAC, or the combination for twenty-four hours. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (E, F) EW8 (E) and TC71 (F) cells were treated with auranofin (5 μM) for twenty-four hours and then labeled with O-propargyl-puromycin for quantification of protein synthesis by flow cytometry. (G) EW8 and TC71 cells were treated with auranofin, NAC, or the combination for either six or twenty-four hours and then labeled with puromycin to quantify protein synthesis. (H) Additional sarcoma cell lines were treated with auranofin, NAC, or the combination for twenty-four hours and then labeled with puromycin for immunoblotting. Protein loading for the immunoblots was normalized using cell number. ****P < 0.0001.
Figure 3
Figure 3
Auranofin reduces the level of the RRM2 protein and blocks DNA replication Ewing sarcoma cells. (A) EW8 and TC71 cells were treated with auranofin, NAC, or the combination for twenty-four hours and then cell lysate was collected for immunoblotting for RRM2. Protein loading for the immunoblots was normalized using cell number. (B) Ewing sarcoma cell lines were treated with auranofin (5 μM), NAC (5 mM), or the combination for 72 hours and then cell viability was quantified using AlamarBlue. Error bars represent the mean ± SD of four technical replicates. The results are representative of two independent experiments. (C) Ewing sarcoma cell lines were treated with auranofin for twenty-four hours and then the cell cycle distribution was analyzed using EdU labeling and propidium iodide. ****P < 0.0001.
Figure 4
Figure 4
Auranofin and reactive oxygen species activate the translational repressor 4E-BP1 in sarcoma cell lines. (A) EW8 and TC71 cells were treated with hydrogen peroxide, NAC, or the combination for three hours and then cell lysates were collected for immunoblotting. Protein loading for the immunoblots was normalized using cell number. (B) EW8 and TC71 cells were treated with auranofin, NAC, or the combination for twenty-four hours and then cell lysates were collected for immunoblotting. Protein loading for the immunoblots was normalized using cell number. (C) TO-4E-BP1-Ala cell lines were treated with doxycycline for 48 hours to induce expression of 4E-BP1-Ala and then cell lysates were collected after labeling the cells with puromycin. (D) Comparison of protein expression (RPPA) in TO-4E-BP1-Ala sarcoma cell lines treated with vehicle or doxycycline for 48 hours. (E) Venn diagram showing the overlap of the RPPA data for proteins that are downregulated by >1.25-fold in the TO-4E-BP1-Ala sarcoma cell lines in the presence of doxycycline.
Figure 5
Figure 5
Auranofin and active 4E-BP1 reduce the level of the PLK1 protein in sarcoma cell lines. (A) TO-4E-BP1-Ala cell lines were treated with doxycycline for 48 hours to induce expression of 4E-BP1-Ala and then cell lysates were collected for immunoblotting. (B) EW8 and TC71 cells were treated with auranofin for either six or twenty-four hours and then cell lysates were collected for immunoblotting. (C) Additional sarcoma cell lines were treated with auranofin, NAC, or the combination for twenty-four hours and then cell lysates were collected for immunoblotting for PLK1. (D) Sarcoma cell lines were treated with the mTORC1/2 inhibitor TAK-228 for twenty-four hours and then cell lysates were collected for immunoblotting. Protein loading for the immunoblots was normalized using cell number.
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