Auranofin radiosensitizes tumor cells through targeting thioredoxin reductase and resulting overproduction of reactive oxygen species

Abstract

Auranofin (AF) is an anti-arthritic drug considered for combined chemotherapy due to its ability to impair the redox homeostasis in tumor cells. In this study, we asked whether AF may in addition radiosensitize tumor cells by targeting thioredoxin reductase (TrxR), a critical enzyme in the antioxidant defense system operating through the reductive protein thioredoxin. Our principal findings in murine 4T1 and EMT6 tumor cells are that AF at 3-10 μM is a potent radiosensitizer in vitro, and that at least two mechanisms are involved in TrxR-mediated radiosensitization. The first one is linked to an oxidative stress, as scavenging of reactive oxygen species (ROS) by N-acetyl cysteine counteracted radiosensitization. We also observed a decrease in mitochondrial oxygen consumption with spared oxygen acting as a radiosensitizer under hypoxic conditions. Overall, radiosensitization was accompanied by ROS overproduction, mitochondrial dysfunction, DNA damage and apoptosis, a common mechanism underlying both cytotoxic and antitumor effects of AF. In tumor-bearing mice, a simultaneous disruption of the thioredoxin and glutathione systems by the combination of AF and buthionine sulfoximine was shown to significantly improve tumor radioresponse. In conclusion, our findings illuminate TrxR in cancer cells as an exploitable radiobiological target and warrant further validation of AF in combination with radiotherapy.

Keywords: ROS; auranofin; buthionine sulfoximine; radiosensitization; thioredoxin reductase.

Figure 1. AF caused apoptosis and cytotoxicity in mouse tumor cells

(A) Tumor cells were treated with AF for 2 h with indicated concentrations, and one day later analyzed for cell viability by MTT assay. Data are shown as mean ± SD (n = 3). (B) Following the same treatments, cell viability was analyzed by an 8-day colony formation assay. Data are shown as mean ± SD (n = 3). (C) Representative scatter plots of apoptosis in 4T1 cells, after AnnexinV/7-AAD staining and assessment by flow cytometry. (D) Summarized data on AF-induced apoptosis in 4T1 and (E) EMT6 tumor cells. Data are shown as mean ± SD (n ≥ 3). One-way ANOVA with Bonferonni's multiple comparison test was used to calculate statistics: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. AF inhibited TrxR and triggered ROS overproduction in tumor cells

(A) TrxR activity was measured by commercial kit and all values were normalized to untreated controls. Data are shown as mean ± SD (n ≥ 3). (B) Representative histogram of intracellular ROS in 4T1 cells, as analyzed by flow cytometry using the CM-H²DCFDA probe. (C–D) Summarized data on ROS production in 4T1 and EMT6 cells pretreated with the ROS scavenger NAC (10 mM) prior to AF. Data are shown as mean ± SD (n = 3). One-way ANOVA with Bonferonni's multiple comparison test was used to calculate statistics: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3. AF radiosensitized aerobic tumor cells and enhanced radiation induced DNA damage

4T1 and EMT6 cells were treated with AF for 2 h at indicated concentrations, while NAC (10 mM) was added 1 h prior and during treatment. (A–B) The radiosensitizing effect of AF was assessed by colony formation assay. Data are shown as mean ± SD (n = 3). (C–D) Counteracting effect of the ROS scavenger NAC at 6 Gy. (E–F) Double-strand DNA breaks were analyzed by flow cytometry using the γH2AX-based foci measurements. Data are shown as mean ± SD (n ≥ 3). One-way ANOVA with Bonferonni's multiple comparison test was used to calculate statistics: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4. AF radiosensitized hypoxic tumor cells

4T1 cells were treated with AF for 2 h at indicated concentrations, while NAC (10 mM) was added 1 h prior and during treatment. To assess hypoxic radiosensitivity, subconfluent cultures were irradiated in a metabolic hypoxia model TMCS. (A–B) Radiosensitizing effect of AF was assessed by colony formation assay. Data are shown as mean ± SD (n = 3). (C–D) Counteracting effect of the ROS scavenger NAC at 10 Gy. Data are shown as mean ± SD (n ≥ 3). One-way ANOVA with Bonferonni's multiple comparison test was used to calculate statistics: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5. AF induced mitochondrial dysfunction in tumor cells

4T1 cells were treated with AF for 2 h at indicated concentrations and the respiratory profiles were examined by a Seahorse Analyzer. (A) Dissection of respiratory rates by the sequential injection of oligomycin, FCCP, rotenone and antimycin A at indicated time points. (B) Summarized data on the baseline respiratory rate, maximum respiratory capacity and ATP turnover. Data are shown as mean ± SD (n = 5). (C–D) Representative measurements of ΔΨm in 4T1 cells by flow cytometry and summarized data on membrane potential. Data are shown as mean ± SD (n = 3). One-way ANOVA with Bonferonni's multiple comparison test was used to calculate statistics: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6. BSO potentiated AF-induced radiosensitization in tumor cells

4T1 cells were exposed to AF and/or BSO at indicated concentrations for 2 and 16 h respectively. (A) Depletion of glutathione by BSO in 4T1 cells. Data are shown as mean ± SD (n ≥ 3). (B) Synergistic cytotoxicity of AF combined with BSO applied at non-cytotoxic concentrations. Data are shown as mean ± SD (n ≥ 3). (C–D) Aerobic and hypoxic radiosensitization by AF combined with BSO, as measured by colony formation assay. Data are shown as mean ± SD (n = 3). (E–F) Counteracting effect of the ROS scavenger NAC under aerobic and hypoxic conditions respectively. Data are shown as mean ± SD (n = 3). One-way ANOVA with Bonferonni's multiple comparison test was used to calculate statistics: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7. AF combined with BSO enhanced the radioresponse of 4T1 tumor in Balb/c mice

AF and BSO were administered subcutaneously for 10 times to tumor-bearing mice, and single dose radiation at 15 Gy was delivered on the second day of treatment. (A) Experimental scheme depicting used treatment protocols. (B) Tumor growth in mice treated with radiation and the combination of AF (3 mg/kg) and BSO (25 mg/kg). (C) Survival curves of mice euthanized at a diameter of 15 mm. (D) Tumor growth in mice treated with radiation and AF only. (E) Tumor growth in mice treated with radiation and BSO only. (F) Assessment of toxicity by body weight loss.

Figure 8. AF combined with BSO enhanced the radioresponse of EMT6 tumor in Balb/c mice

AF and BSO were administered as above in 4T1 tumor model, and single dose radiation at 12 Gy was delivered on the second day of treatment. (A) Tumor growth of mice treated with radiation and the combination of AF (3 mg/kg) and BSO (25 mg/kg). (B) Survival curves of mice euthanized at diameter of 15 mm. (C) Assessment of toxicity by body weight loss.