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. 2023 Jan 19;14(1):42.
doi: 10.1038/s41419-023-05586-6.

Dual inhibition of thioredoxin reductase and proteasome is required for auranofin-induced paraptosis in breast cancer cells

Min Ji Seo #  1   2 , In Young Kim #  1   2   3 , Dong Min Lee  1   2 , Yeon Jung Park  1   2 , Mi-Young Cho  1 , Hyo Joon Jin  1   4 , Kyeong Sook Choi  5   6
Affiliations

Dual inhibition of thioredoxin reductase and proteasome is required for auranofin-induced paraptosis in breast cancer cells

Min Ji Seo et al. Cell Death Dis. .

Abstract

Auranofin (AF), a gold (I)-containing phosphine compound, is being investigated for oncological application as a repurposed drug. We show here that 4~5 µM AF induces paraptosis, a non-apoptotic cell death mode characterized by dilation of the endoplasmic reticulum (ER) and mitochondria, in breast cancer cells. Although the covalent inhibition of thioredoxin reductase (TrxR), an enzyme that critically controls intracellular redox homeostasis, is considered the primary mechanism of AF's anticancer activity, knockdown of TrxR1 did not induce paraptosis. Instead, both TrxR1 knockdown plus the proteasome inhibitor (PI), bortezomib (Bz), and 2 μM AF plus Bz induced paraptosis, thereby mimicking the effect of 5 μM AF. These results suggest that the paraptosis induced by 5 μM AF requires the inhibition of both TrxR1 and proteasome. We found that TrxR1 knockdown/Bz or subtoxic doses of AF and Bz induced paraptosis selectively in breast cancer cells, sparing non-transformed MCF10A cells, whereas 4~5 μM AF killed both cancer and MCF10A cells. GSH depletion was found to be more critical than ROS generation for the paraptosis induced by dual TrxR1/proteasome inhibition. In this process, the ATF4/CHAC1 (glutathione-specific gamma-glutamylcyclotransferase 1) axis leads to GSH degradation, contributing to proteotoxic stress possibly due to the accumulation of misfolded thiol-containing proteins. These results suggest that the paraptosis-inducing strategy of AF plus a PI may provide an effective therapeutic strategy against pro-apoptotic therapy-resistant cancers and reduce the potential side effects associated with high-dose AF.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Auranofin induces paraptosis in several breast cancer cell lines.
A–D Cells were treated with the indicated concentrations of AF or CDDO-ME for 24 h. E, F MDA-MB 435 S cells pretreated with the indicated doses of z-VAD-fmk (z-VAD), necrostatin-1 (Nec), ferrostatin-1 (Fer), 3-methyladenine (3-MA), bafilomycin A1 (Baf), or cycloheximide (CHX) were further treated with 5 μM AF for 24 h. A, E Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.01. B, C, F Cellular morphologies were observed by phase-contrast microscopy. White and black arrow heads denote blebbing and apoptotic body, respectively (C). Bars, 20 μm. D Western blotting of caspase-3 and PARP was performed using β-actin as a loading control. G YFP-ER cells treated with 5 μM AF and/or 2 μM CHX for 24 h were stained with MitoTracker-Red (MTR). Cells were observed by confocal microscopy. Bars, 20 μm. H MDA-MB 435 S cells were treated with the indicated concentrations of AF for 12 h, and Western blotting of the indicated proteins was performed using β-actin as a loading control. I, J MDA-MB 435 S cells pretreated with the indicated inhibitors were further treated with 5 μM AF for 24 h. I Cellular viability was assessed using IncuCyte. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. J Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm.
Fig. 2
Fig. 2. TrxR1/proteasome inhibition is required for AF-induced paraptosis.
A, B MDA-MB 435 S cells were transfected with the negative control siRNA (siNC) or three different siRNAs against TrxR1 (siTrxR1) for 24 h. A Knockdown of TrxR1 was confirmed by Western blotting with β-actin used as a loading control (left). Cellular viability in transfected cells was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test (right). B Morphologies of the transfected cells were observed by phase-contrast microscopy. Bars, 20 μm. C MDA-MB 435 S cells were treated with the indicated concentrations of AF (left) or Bz (right) for 12 h. Western blotting of the ubiquitinated proteins was performed using β-actin as a loading control. D MDA-MB 435 S cells transfected with UbG76V-GFP were treated with the indicated concentrations of AF or Bz for 12 h. The fluorescence intensity was assessed, as described in the Materials and Methods. E TrxR1 activity assay was performed in MDA-MB 435 S cells treated with AF and/or Bz at the indicated concentrations for 20 min as described in Materials and Methods (left). Rat liver TrxR was used as a positive control (Pos. cont.). The percentage of TrxR activity was normalized to that of untreated cells (100%). Data represent the means ± SEM. (n = 3). One way-ANOVA and Dunn’s test. *p < 0.001. Changes in TrxR1 protein levels following treatment with 2.5 or 5 nM Bz were examined by Western blotting usig β-actin as a loading control (right). F, G MDA-MB 435 S cells were transfected with siNC or siTrxR1 for 24 h and further treated with the indicated doses of Bz or Cfz. H, I MDAS-MB 435 S cells transfected with siNC or siTrxR1 were further treated with the indicated concentrations of AF for 24 h. J, K MDA-MB 435 S cells were transfected with siNC or siTrxR1 for 24 h, pretreated with the indicated doses of z-VAD, Nec, Fer, 3-MA, Baf, or CHX, and further treated with 5 nM Bz for 24 h. F, H, J Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.001 (F), *p < 0.05 (H), *p < 0.05 (J). Cellular morphologies were observed by phase-contrast microscopy (G, L) or employing IncuCyte (I). Bars, 20 μm. K YFP-ER cells transfected with siNC or siTrxR1 were further treated with 5 nM Bz and/or 2 μM CHX for 24 h and then stained with MTR. Cells were observed by confocal microscopy. Bars, 20 μm.
Fig. 3
Fig. 3. Non-transformed cells are killed by 4~5 μM AF, but spared by TrxR1 knockdown plus Bz or 2 μM AF plus Bz.
A, B MCF10A cells were treated with the indicated concentrations of AF for 24 h. C, D MCF10A cells pretreated with the indicated doses of z-VAD, Nec, Fer, 3-MA, Baf, or CHX were further treated with AF for 24 h. E, F MCF10A cells transfected with siNC or siTrxR1 for 24 h were further treated with the indicated doses of Bz for 24 h. G, H MCF10A cells were treated with the indicated concentrations of AF and/or Bz for 24 h. A, C, E, G Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. * p < 0.001; ns (non-significant). B, D, F, H Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm.
Fig. 4
Fig. 4. Combining low doses of AF and PI induces paraptosis in breast cancer cells.
A–C Cells were treated with the indicated concentrations of AF and/or PIs for 24 h. D, E MDA-MB 435 S cells pretreated with the indicated dose of z-VAD, Nec, Fer, 3-MA, Baf, or CHX were further treated with 2 μM AF plus 5 nM Bz for 24 h. A, D Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. B. B Isoboles for the combinations of AF and Bz or AF and Cfz that proved iso-effective (IC50) for inhibiting cell viability in the tested cancer cells. C, E Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. F YFP-ER cells treated with 2 μM AF plus 5 nM Bz and/or 2 μM CHX for 24 h were stained with MTR. Cells were observed by confocal microscopy. Bars, 20 μm.
Fig. 5
Fig. 5. Disruption of thiol homeostasis rather than ROS generation is critical for the paraptosis induced by inhibition of TrxR1 and proteasome.
A MDA-MB 435 S cells were treated with the indicated concentrations of AF and/or Bz for the indicated durations. Treated cells were incubated with CM-H2DCF-DA (DCF-DA) and subjected to flow cytometry. Cells treated with 10 mM H2O2 for 30 min were used as the positive control. Data are expressed as the fold change in DCF fluorescence intensity in treated cells compared to that in untreated cells. Data represent the means ± SD. (n = 3). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.005. B, C MDA-MB 435 S cells were pretreated with the indicated doses of N-acetylcysteine (NAC), glutathione reduced ethyl ester (GEE), N-(2-mercapto-propionyl)-glycine (NMPG), tiron, ascorbic acid (AA), Cu(II)(3,5-diisopropylsalicylate)2 (CuDIPs), or manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP; MnT) and further treated with 20 nM Cfz plus 2 μM AF for 24 h. D, E MDA-MB 435 S cells transfected with siNC or siTrxR1 were treated with the indicated doses of NAC, GEE, CuDIPs, or MnT and further treated with 5 nM Bz for 24 h. B, D Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. C, E Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. F MDA-MB 435 S cells transfected with siNC or siTrxR1 were further treated with 5 nM Bz for 12 h (left) or treated with 5 nM Bz plus 2 μM AF for 12 h (right). G MDA-MB 435 S cells transfected with siNC or siTrxR1 were treated with 2 mM NAC and/or 5 nM Bz for 12 h, or treated with 2 mM NAC and/or AF plus Bz at the indicated concentrations for 12 h. F, G GSH levels were assessed, as described in the Materials and Methods.
Fig. 6
Fig. 6. AF-mediated TrxR1 inhibition enhances Bz-induced ER stress, and ATF4 plays a critical role in the paraptosis induced by TrxR1/proteasome inhibition.
A MDA-MB 435 S cells transfected with siNC or siTrxR1 were treated with 5 nM Bz for 12 h, or with the indicated concentrations of Bz and/or AF for 12 h. B, D MDA-MB 435 S cells transfected with siNC, ATF4-targeting siRNA (siATF4), or CHOP-targeting siRNA (siCHOP) were transfected with TrxR1 and further treated with 5 nM Bz for 24 h, or treated with the indicated concentrations of Bz and/or AF for 24 h. A, B Western blotting of the indicated proteins was performed using β-actin as a loading control. C Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. D Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm.
Fig. 7
Fig. 7. ATF4/CHAC1 critically contributes to the paraptosis induced by TrxR1/proteasome inhibition by degrading GSH.
A MDA-MB 435 S cells were treated with the indicated concentrations of AF alone (left) or Bz and/or AF (right) for 12 h. MDA-MB 435 S cells transfected with siNC or siTrxR1 were treated with 5 nM Bz for 12 h (middle). MDA-MB 435 S cells transfected with siNC, siATF4, or CHAC1-targeting siRNA (siCHAC1) were cotransfected with TrxR1 and further treated with 5 nM Bz for 24 h (B–E) or 12 h (F), or treated with the indicated concentrations of Bz and/or AF for 24 h (B–E) or 12 h (F). G, H MDA-MB 435 S cells pretreated with 2 μM CHX or 2 mM NAC were treated with 2 μM AF plus 5 nM Bz for 12 h. I Cells were treated with 5 nM Bz and/or 2 μM AF for 12 h. A, C, H, I Western blotting of the indicated proteins was performed using β-actin as a loading control. B The mRNA levels of ATF4 or CHAC1 were assessed by qRT-PCR. D Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. E Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. F, G GSH levels were assessed, as described in Materials and Methods.
Fig. 8
Fig. 8. Hypothetical model for the mechanism underlying the paraptosis induced by auranofin or simultaneous inhibition of TrxR1 and proteasome.
For the induction of auranofin-induced paraptosis, both TrxR1 inhibition and proteasome inhibition are required. Simultaneous TrxR1/proteasome inhibition triggers the ISR due to proteotoxic stress. In this process, GSH degradation, which is mediated by the ATF4/CHAC1 axis, critically contributes to inducing paraptosis by aggravating proteotoxic stress.
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