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. 2025 Apr 15;23(3):377–387. doi: 10.1111/vco.13062

Auranofin Induces ER Stress‐Mediated Apoptosis, and Its Combination With Bortezomib Elicits Paraptosis‐Like Cell Death in Malignant Canine Mammary Tumour Cells

Yoon‐Ho Suh 1, Se‐Hoon Kim 1, Ki‐Hoon Song 2, Jun‐Yeol Choi 1, Min‐Ok Ryu 1, Robert B Rebhun 3, Kyoung‐Won Seo 1,✉
PMCID: PMC12378087  PMID: 40235190

ABSTRACT

Canine mammary tumours (CMT) are common in female dogs, often associated with malignancy and limited responses to conventional therapies. This study explores the potential of Auranofin (AF) in malignant CMT, focusing on its ability to induce distinct cell deaths. AF inhibited thioredoxin reductase (TrxR) activity, cell proliferation, and colony formation across malignant CMT cell lines, demonstrating significant anticancer effects. In AF‐sensitive cell lines (CMT‐U27, CHMm, and CHMp), 0.5–2 μM AF induced endoplasmic reticulum (ER) stress‐mediated apoptosis, while concentrations above 3 μM caused near‐complete cell death via additional proteasome inhibition. However, in AF‐resistant cell lines (CIPp and CIPm), AF concentrations required for near‐complete cell death were higher, expected to be challenging to achieve clinically. Therefore, we combined sublethal doses of AF (~2 μM) with the proteasome inhibitor Bortezomib (Bz) in these cells. The combination exhibited synergistic cytotoxicity and induced extensive cytoplasmic vacuolation. Live‐cell staining revealed the ER origin of vacuoles, and cycloheximide pretreatment inhibited both vacuolation and AF + Bz‐induced cell death, indicating features of paraptosis. While apoptosis could not be excluded, it was classified as paraptosis‐like cell death occurring concurrently with apoptosis. Further analysis supported that this cell death is related to enhanced ER stress from AF‐induced TrxR inhibition and Bz‐induced proteasome inhibition. Based on these findings, we propose AF alone or combined with Bz as a promising therapeutic strategy for malignant CMT. Our findings highlight AF's potential to induce ER stress‐mediated apoptosis and paraptosis‐like cell death in canine cancer cells, expanding therapeutic options for targeting cancers in dogs.

Keywords: Auranofin, canine mammary tumour, endoplasmic reticulum stress, paraptosis, proteasome inhibitor, thioredoxin reductase

1. Introduction

Canine mammary tumours (CMT) are the most common tumours in female dogs [1], with malignancy and prognosis influenced by reproductive status [2, 3]. Although early ovariohysterectomy reduces CMT risk, nearly half of the cases in intact females are malignant and late‐spayed dogs face a similar risk of developing malignant tumours as intact females [3]. These malignancies pose significant therapeutic challenges. Standard treatments including surgery and chemotherapy are often limited in effectiveness, especially against malignant tumours that are resistant or recur [4, 5]. Moreover, conventional chemotherapy with cytotoxic agents is commonly associated with adverse effects, including gastrointestinal disturbances, bone marrow suppression, and systemic toxicity, all of which can impact the patient's quality of life [6]. This highlights the need for new therapeutic approaches in veterinary oncology that can effectively target treatment‐resistant malignant CMT with minimal side effects.

Drug repurposing involves identifying new therapeutic uses for existing drugs beyond their original medical indications [7]. This strategy is a promising approach to accelerate the development of new treatments, as it leverages known safety profiles and clinical data from approved drugs [8]. Auranofin (AF) is an FDA‐approved drug initially used for human rheumatoid arthritis [9]. In veterinary medicine, AF is used to treat idiopathic polyarthritis and pemphigus complex in dogs and cats, with oral doses of 0.05–0.3 mg/kg every 12 h [10]. Recently, AF has demonstrated significant anticancer effects both in vitro and in vivo in various human cancers, including chronic lymphocytic leukaemia (CLL) [11] and osteosarcoma [12]. In addition, AF has shown in vitro efficacy against classical Hodgkin lymphoma [13], non‐small cell lung cancer [14], and breast cancer [15]. AF's anticancer effects are linked to its ability to inhibit thioredoxin reductase (TrxR), an enzyme that helps maintain redox balance in cancer cells [16, 17]. By inhibiting TrxR, AF increases oxidative stress, leading to stress in the endoplasmic reticulum (ER) and cell death [11, 18]. Given its demonstrated effects on human breast cancer and established use in animals, AF warrants further investigation into its anticancer potential in CMT.

Paraptosis is a distinct mode of programmed cell death different from apoptosis, characterised by large vacuoles in the cytoplasm originating from a swollen ER or mitochondria [19, 20, 21]. Since paraptosis is not inhibited by standard apoptotic blockers, it could serve as an effective alternative cell death pathway in cancers resistant to apoptosis‐driven therapies [20, 21]. In human breast cancer cells, AF has been shown to induce paraptosis through dual inhibition of TrxR and the proteasome [22]. However, to our knowledge, paraptosis has not been described in canine cancer cells.

Recent research has examined AF's effects on CMT cells, demonstrating its ability to induce apoptosis via the PI3K/AKT pathway [23]. While this study provided valuable insights, it primarily focused on apoptosis and did not explore alternative mechanisms of cell death. Therefore, this study aims to investigate the effects of AF on CMT cells, with a focus on its potential to suppress TrxR activity, induce ER stress‐mediated apoptosis, and paraptosis‐like cell death.

2. Materials and Methods

2.1. Cell Culture and Cell Line Authentication Statement

Five malignant CMT cell lines (CIPp, CIPm, CHMp, CHMm, and CMT‐U27) were used in this study. CIPp, CIPm, CHMp, and CHMm were derived from primary or metastatic lesions of two malignant CMT patient dogs admitted to the Veterinary Hospital at The University of Tokyo [24], and kindly provided by Dr. Takayuki Nakagawa (Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo). CMT‐U27 cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were screened for mycoplasma contamination, and their canine origin was confirmed by genomic DNA species verification. Cells were cultured in Roswell Park Memorial Institute (RPMI)‐1640 medium (Solbio, Suwon, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; T&I, Chuncheon, Republic of Korea) and 100 U/mL penicillin–streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained in a humidified incubator at 37°C with 5% CO2. Cell morphologies were observed using a Leica DMi1 phase‐contrast microscope (Leica Microsystems, Wetzlar, Germany).

2.2. Drugs and Treatment

AF (Sigma Aldrich, St. Louis, MO, USA), Bortezomib (Bz; MedChemExpress, Monmouth Junction, NJ, USA), and Cycloheximide (CHX; Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich) to prepare stock solutions and diluted in cell culture medium to achieve working concentrations. The final DMSO concentration in drug‐treated cells was maintained below 0.125%. Cells treated with culture medium containing 10% FBS and antibiotics served as controls. CHX was applied 2 h prior to the addition of other drugs or medium, as described previously [22].

2.3. Cell Viability Assay

Two thousand or five thousand cells were seeded in a 96‐well plate (SPL Life Sciences, Pocheon, Republic of Korea) and stabilised for 24 h. Then, cells were treated as indicated for 48 h. Viability was assessed using the Cell Counting Kit‐8 (CCK‐8; Dojindo Laboratories, Kumamoto, Japan), which utilises water‐soluble tetrazolium salt‐8 (WST‐8). For CHX experiments, cells were treated for 24 h, then replaced with drug‐free medium for an additional 24 h to mitigate CHX's inhibitory effect on cellular metabolism. Subsequently, 10 μL of CCK‐8 solution was added, and optical density was measured at 450 nm using a microplate reader (BioTek ELx800; BioTek Instruments, Winooski, VT, USA) after a 2‐h incubation at 37°C with 5% CO2. Absorbance of control groups was set to 100%, and relative viability was calculated.

2.4. Clonogenic Assay

Cells were seeded in 6‐well plates (SPL Life Sciences) at densities of 200–3000 cells/well and stabilised for 24 h. Cells were then treated with the indicated AF concentrations for 48 h, followed by replacement with drug‐free medium and incubation at 37°C with 5% CO2 for 5 days. The medium was refreshed 3 days after the initial replacement. Colonies were fixed and stained with 0.5% crystal violet (Sigma Aldrich) in methanol for 30 min, then imaged. To quantify colony formation, 2 mL of methanol was added per well, and plates were shaken for 20 min to dissolve the stain. Optical density at 570 nm was measured, with the control group's absorbance set to 100% for relative calculations.

2.5. Protein Extraction

A total of 1 × 106 cells were seeded in 90 mm dishes (SPL Life Sciences) and treated under the specified conditions for 24 or 48 h. After washing with cold phosphate‐buffered saline (PBS; Welgene, Gyeongsan, Republic of Korea), cells were lysed using cell lysis buffer (Smartgene, Daejeon, Republic of Korea). Lysates were incubated on ice for 30 min and centrifuged at 12 000 rpm (15 928 × g) at 4°C for 20 min. Supernatants were collected, and protein concentrations were measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific).

2.6. TrxR Activity Assay

TrxR activity was measured using a TrxR colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) based on its ability to utilise NADPH to reduce 5,5′‐dithio‐bis (2‐dinitrobenzoic acid) (DTNB) to 5‐thio‐2‐nitrobenzoic acid (TNB), yielding a yellow product. Measurement of TrxR activity in the presence and absence of a TrxR inhibitor enabled the correction of non‐TrxR‐dependent DTNB reduction. Equal amounts of total protein (35 μg) were added to each well of a 96‐well plate. Reactions were initiated by adding a master mix containing DTNB and NADPH to each well [25]. The plate was gently shaken for 10 s, and absorbance at 450 nm was measured. All samples and controls were assayed in triplicate, and TrxR activity was calculated using a formula provided in the kit protocol.

2.7. Western Blot Analysis

Equal amounts of protein were separated on 10%–12% SDS‐PAGE gels and transferred to Clear Blot Membrane‐N (ATTO, Tokyo, Japan). The membranes were blocked with EveryBlot Blocking Buffer (Bio‐Rad, Hercules, CA, USA) for 5 min or with bovine serum albumin (BSA; Georgiachem, Chuncheon, Republic of Korea) in TBST (Tris‐buffered saline and Tween 20) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with primary antibodies (Tables S1 and S2). The specificity of each primary antibody was assessed in canine cell lysates (Figure S1). After washing with TBST, the membranes were incubated for 1 h at room temperature with secondary antibodies, including horseradish peroxidase (HRP)‐conjugated anti‐mouse antibody (1:15 000 dilution; ABclonal Technology, Wuhan, China) and HRP‐conjugated anti‐rabbit antibody (1:15 000 dilution; ABclonal Technology). Protein bands were visualised using ECL High Femto Solution (Smartgene) and detected with an ImageQuant LAS 4000 system (GE Healthcare, Buckinghamshire, UK).

2.8. Drug Synergism Analysis

To evaluate the synergistic effects of AF and Bz on AF‐resistant CMT cell lines, we utilised the SynergyFinder web application (version 3.10.3; available at https://www.synergyfinderplus.org/). Cell viability data from the AF and Bz combinations were obtained after 48 h of treatment. These data were then uploaded to the application, and the Loewe synergy model was applied to calculate the synergy scores.

2.9. Live‐Cell Staining of the ER and Nucleus

After treatments, cells were stained with 200 nM ER‐Tracker Green (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 20 min and washed with PBS. Then, 2 μg/mL Hoechst 33342 (BLD pharm, Shanghai, China) was added and incubated for 10 min. After washing with PBS, cells were visualised using an EVOS FL Cell Imaging System (Thermo Fisher Scientific).

2.10. Statistical Analysis

All statistical analyses were performed using GraphPad Prism (version 10.1.2, GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM). Statistical methods were selected based on data normality and homogeneity of variances. Normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated using Brown–Forsythe's test. Non‐normally distributed data were analysed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. For normally distributed data, one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied when variances were homogeneous, and Welch's ANOVA followed by Dunnett's T3 multiple comparisons test was used when variances were unequal. *p < 0.05 was considered statistically significant.

3. Results

3.1. AF Inhibits Proliferation, Colony Formation, and TrxR Activity in CMT Cells

Five malignant CMT cell lines were treated with increasing AF concentrations for 48 h to evaluate the anticancer effects of AF, and cell viability was assessed. Dose–response curves were plotted to determine IC50 values for each cell line (Figure 1A). AF effectively suppressed cell proliferation, with varying IC50 values observed across the cell lines. CMT‐U27, CHMm, and CHMp exhibited relatively low IC50 values of 0.9116, 1.499, and 1.748 μM, respectively. In contrast, CIPm and CIPp showed higher IC50 values of 3.247 and 3.870 μM. An IC50 threshold of 2 μM was used to classify the cell lines. Based on this threshold, CMT‐U27, CHMm, and CHMp were categorised as AF‐sensitive, whereas CIPp and CIPm were classified as AF‐resistant. In AF‐sensitive cell lines, significant viability reductions were observed at concentrations of 0.78–1.56 μM (Figure 1B), while AF‐resistant cell lines showed significant decreases starting at 1.56–3.13 μM (Figure 1C). Clonogenic assays revealed dose‐dependent inhibition of colony formation across all cell lines (Figure 1D), with substantial reductions at 0.5 μM and near‐complete suppression at 1 μM in most cell lines. Inhibition was more pronounced in AF‐sensitive cell lines compared to AF‐resistant ones. Previous studies reported that AF exhibits anticancer effects by inhibiting TrxR activity [16, 17]. To assess the effect of AF on TrxR activity, TrxR assays were performed on representative AF‐sensitive (CHMp) and AF‐resistant (CIPp) cell lines. AF treatment resulted in a significant, dose‐dependent reduction in TrxR activity in both cell lines, with greater inhibition observed in CHMp than in CIPp (Figure 1E). Together, these results confirmed the anticancer effects of AF in CMT cells, with varying responsiveness among different cell lines.

FIGURE 1.

FIGURE 1

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Auranofin (AF) suppresses cell proliferation, colony formation, and thioredoxin reductase (TrxR) activity in canine mammary tumour (CMT) cell lines. (A) Dose–response curves showing half‐maximal inhibitory concentration (IC50) values for five CMT cell lines treated with increasing concentrations of AF for 48 h. (B, C) Cell viability of AF‐sensitive (B) and AF‐resistant (C) CMT cell lines following 48‐h AF treatment (n = 16–18). (D) Colony formation assay demonstrating dose‐dependent inhibition of colony formation across five CMT cell lines treated with AF. Colony formation was quantified by dissolving crystal violet stain in methanol, measuring the optical density at 570 nm, and normalising values to the control group. (n = 3–5) (E) TrxR activity assay showing dose‐dependent inhibition of TrxR activity by AF in representative AF‐sensitive and AF‐resistant CMT cells (n = 3). Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, compared to the control group of each cell line.

3.2. AF Induces ER Stress, Proteasome Inhibition, and Apoptosis in AF‐Sensitive CMT Cells

To examine AF's mechanisms in AF‐sensitive CMT cell lines, CMT‐U27, CHMm, and CHMp cells were treated with increasing AF concentrations, and the expression of proteins associated with ER stress, proteasome inhibition, and apoptosis was analysed. Western blot analysis (Figure 2) revealed a concentration‐dependent ER stress response, with IRE1α expression rising up to 2 μM AF, followed by ATF4 induction at higher concentrations. Proteasome inhibition was indicated by the accumulation of ubiquitinated proteins, noticeable at 3 μM and more pronounced at 4 μM. Additionally, a dose‐dependent decrease in pro‐caspase‐3 and increases in cleaved PARP and Bax suggest AF induces apoptosis in these cell lines. In summary, 0.5–2 μM AF effectively induced ER stress‐mediated apoptosis in AF‐sensitive cells, while concentrations exceeding 3 μM led to near‐complete cell death through additional proteasome inhibition. These results indicated that AF induces anticancer effects in AF‐sensitive CMT cells through ER stress, proteasome inhibition, and apoptosis.

FIGURE 2.

FIGURE 2

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Auranofin (AF) induces endoplasmic reticulum (ER) stress, inhibits proteasome function, and triggers apoptosis in AF‐sensitive canine mammary tumour (CMT) cell lines. AF‐sensitive cell lines (CMT‐U27, CHMm, and CHMp) were treated with increasing concentrations of AF for 48 h, and protein expression levels were assessed by Western blot analysis. CPARP, cleaved PARP; Procas‐3, pro‐caspase‐3; Ub‐proteins, ubiquitinated proteins.

3.3. Combination of Sub‐Lethal Doses of AF and Bz Exhibits Synergistic Cytotoxicity in AF‐Resistant CMT Cells

In AF‐sensitive CMT cell lines, AF concentrations above 3 μM induced proteasome inhibition (Figure 2) and coincided with near‐complete cell death (Figure 1B), suggesting a critical role for proteasome inhibition in AF‐induced cytotoxicity. However, the AF concentrations required to achieve near‐complete cell death in resistant cell lines are considered clinically unattainable in dogs' plasma with current oral dosing protocols. To enhance cytotoxicity at lower AF concentrations, we combined sublethal AF doses (≤ 2 μM) with the proteasome inhibitor Bz in AF‐resistant cell lines (CIPp and CIPm) to mimic the mechanism observed with AF concentrations exceeding 3 μM in AF‐sensitive cell lines. Combination of AF and Bz reduced cell viability compared to either agent alone, demonstrating enhanced cytotoxicity (Figure 3A,C). The positive values of Loewe synergy score also supported this conclusion. The combination revealed widespread synergy, with the highest scores observed at AF 2 μM and Bz 16.7 nM in both cell lines (Figure 3B,D). Therefore, this concentration combination was selected for further experiments.

FIGURE 3.

FIGURE 3

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Combination of Auranofin (AF) and Bortezomib (Bz) synergistically enhances cell death in AF‐resistant canine mammary tumour (CMT) cell lines. Cell viability was measured in AF‐resistant CMT cell lines (CIPp and CIPm) treated with various concentration combinations of AF and Bz for 48 h. In each cell line, the left heatmaps (A, C) show cell viability across different concentration combinations, and the right heatmaps (B, D) display Loewe synergy scores. Positive synergy scores (shaded in red) indicate synergistic interactions and negative scores (shaded in green) represent antagonistic effects. Data represent three independent experiments.

3.4. Combination of AF and Bz Induces Paraptosis‐Like Cell Death in AF‐Resistant CMT Cells

When observing morphological changes over time in AF‐resistant CMT cell lines (CIPp and CIPm) treated with 2 μM AF and 16.7 nM Bz (Figure 4), small cytoplasmic vacuoles appeared in some cells after 8 h. By 24 h, most cells exhibited extensive cytoplasmic vacuolization, and vacuoles became larger at 32 h. Eventually, cells began to detach and float with intact vacuoles inside.

FIGURE 4.

FIGURE 4

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Combination of 2 μM Auranofin (AF) and 16.7 nM Bortezomib (Bz) induces extensive cytoplasmic vacuolation in AF‐resistant canine mammary tumour (CMT) cell lines. Morphological changes in AF‐resistant CMT cell lines (CIPp and CIPm) were observed at various time points following the combination treatment. Representative images were captured using phase‐contrast microscopy. For each cell line, the lower images represent a 4× magnified view of the white boxes marked in the corresponding upper images. Scale bar, 100 μm.

Massive cytoplasmic vacuolization is a hallmark of paraptosis, a form of non‐apoptotic cell death that requires active protein synthesis [19, 20]. To determine whether the observed vacuolation was indicative of paraptosis, we pretreated AF‐resistant CMT cell lines with CHX, a protein synthesis inhibitor known to prevent paraptosis [19]. Vacuolation was absent with AF alone and occurred in less than 20% of cells treated with Bz alone, with random analysis of over 300 cells showing vacuoles in 16.8% of CIPp and 5.8% of CIPm cells after 24 h (Figure 5A). In contrast, the combination (AF + Bz) induced extensive vacuolation in nearly all cells, while CHX pretreatment completely inhibited vacuolation and partially restored cell viability (Figure 5B). These findings support a paraptosis‐like mechanism and a potential association connecting vacuole formation with reduced cell viability.

FIGURE 5.

FIGURE 5

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Cycloheximide (CHX) pretreatment prevents cytoplasmic vacuolation and reduces cytotoxicity induced by the combination of Auranofin (AF) and Bortezomib (Bz) in AF‐resistant canine mammary tumour (CMT) cell lines. (A) Morphological changes in AF‐resistant CMT cell lines (CIPp and CIPm) were assessed by phase‐contrast microscopy after treatment with 2 μM AF, 16.7 nM Bz, or the combination of AF and Bz (AF + Bz) for 24 h, with or without CHX pretreatment. Scale bars represent 50 μm. (B) Cell viability was measured to determine the effect of CHX on AF + Bz‐induced cytotoxicity. Cells were pretreated with CHX (2 μM) or left untreated for 2 h, then treated with AF + Bz for 24 h. Data are shown as mean ± SEM (n = 18). The y‐axis includes a break (indicated by two parallel slanted lines) to exclude the range of cell viability (%) from 55 to 90 for improved visualisation. Statistical significance is indicated as ***p < 0.001, with only the relevant comparison included for clarity.

Paraptosis is characterised by vacuoles originating from a swollen ER or mitochondria [19]. To trace the origin of the vacuoles observed in AF + Bz‐treated cells, we stained the cells with ER‐Tracker Green and Hoechst 33342. Live‐cell staining revealed the vacuoles primarily co‐localised with the ER (Figure 6), suggesting the AF + Bz‐induced vacuoles originate from ER swelling and indicating a likely involvement of ER stress in this process.

FIGURE 6.

FIGURE 6

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Cytoplasmic vacuolation induced by the combination of Auranofin (AF) and Bortezomib (Bz) originates from the endoplasmic reticulum (ER). CIPm cells were treated with 2 μM AF and 16.7 nM Bz for 24 h, and then subjected to live‐cell staining. The ER in each cell was stained with ER‐Tracker Green, and the nuclei were stained with Hoechst 33342. Representative images were acquired using a fluorescence microscope. Scale bar, 25 μm.

Collectively, AF + Bz‐induced cell death exhibits features of paraptosis: (1) extensive vacuolation, (2) ER‐originating vacuoles, and (3) suppression of vacuolation and cytotoxicity by CHX. However, cleaved PARP expression (Figure S2) suggests concurrent apoptosis, as also observed in sensitive cell lines (Figure 2). Thus, this cell death is classified as paraptosis‐like rather than typical paraptosis.

3.5. AF + Bz Induces Paraptosis‐Like Cell Death via TrxR Inhibition, Proteasome Inhibition, and ER Stress

To elucidate the mechanism underlying AF + Bz‐induced paraptosis‐like cell death in AF‐resistant CMT cells, TrxR activity assays and Western blot analyses were performed. Consistent with previous findings (Figure 1E), 2 μM AF effectively inhibited TrxR activity (Figure 7A). AF + Bz inhibited TrxR activity to a level similar to AF alone at 2 μM, showing that TrxR inhibition is primarily driven by AF, while Bz alone has no effect on TrxR activity. In contrast, Bz alone or combined with AF increased ubiquitinated protein levels, highlighting its role in proteasome inhibition, whereas 2 μM AF had no effect on proteasome activity in AF‐resistant cell lines (Figure 7B). AF + Bz also elevated IRE1α expression more than either agent alone (Figure 7B), suggesting that the combination induces ER stress more effectively than single‐agent treatments. In cells pretreated with CHX, the AF + Bz‐induced increases in both IRE1α and ubiquitinated proteins were suppressed. These findings demonstrate that CHX prevents cytoplasmic vacuolation and restores cell viability by reducing proteasome dysfunction and ER stress. Collectively, these results indicate the AF + Bz combination induces paraptosis‐like cell death by inhibiting TrxR activity through AF and proteasome activity through Bz, leading to ER stress of CMT cells. This highlights that combining AF with Bz in resistant cell lines effectively mimics the impact of lethal doses (≥ 3 μM) of AF in sensitive cell lines, resulting in pronounced cancer cell death.

FIGURE 7.

FIGURE 7

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Combination of Auranofin (AF) and Bortezomib (Bz) suppresses thioredoxin reductase (TrxR) and proteasome activity, and induces endoplasmic reticulum (ER) stress in AF‐resistant canine mammary tumour (CMT) cells. (A) TrxR activity was measured in CIPp cells treated with 2 μM AF, 16.7 nM Bz, or the combination for 24 h. Data are presented as mean ± SEM (n = 3). Statistical significance is indicated as **p < 0.01, compared to the non‐treated control group. (B) Western blot analysis was performed on AF‐resistant cells treated as indicated for 24 h, with or without 2‐h pretreatment with 2 μM Cycloheximide (CHX). Ub‐proteins, ubiquitinated proteins.

4. Discussion

This study provides new insights into the cytotoxic mechanisms of AF in malignant CMT cells, expanding beyond previous research that primarily focused on apoptosis. In veterinary oncology, AF's anticancer effects have been largely associated with apoptosis, with an in vitro study in canine lymphoma [26], in vitro and xenograft models of osteosarcoma [27], and a clinical pilot study in dogs with osteosarcoma [28]. Similarly, a recent study revealed AF's role in inducing apoptosis in CMT cells but did not examine other mechanisms [23]. In contrast, our study expands this understanding by exploring the multifaceted mechanisms underlying AF's cytotoxic effects, including TrxR inhibition, proteasome inhibition, ER stress, and paraptosis‐like cell death.

TrxR is critical for maintaining cellular redox balance by reducing thioredoxin (Trx), which regulates reactive oxygen species (ROS) generated during metabolism [29]. In cancer cells, high metabolic demands increase reliance on TrxR for redox homeostasis [30]. Our study demonstrated that AF‐sensitive CMT cell lines showed greater reductions in TrxR activity at lower AF concentrations, likely contributing to their heightened sensitivity to AF. In contrast, AF‐resistant CMT cell lines exhibited resilience to TrxR inhibition, potentially due to compensatory redox pathways. This aligns with findings in human lung cancer cells, where AF resistance is associated with alternative mechanisms such as glutathione reductase (GSR) and NAD(P)H quinone dehydrogenase 1 (NQO1), which help mitigate oxidative stress in resistant cells [31].

The proteasome is essential for maintaining cellular proteostasis by degrading misfolded or damaged proteins [32]. Proteasome inhibition results in the accumulation of ubiquitinated proteins, inducing proteotoxic stress and activating ER stress pathways such as the unfolded protein response (UPR) and integrated stress response (ISR) [33]. In human breast cancer cells, 5 nM Bz has been shown to cause similar levels of ubiquitinated protein accumulation as observed with 4–5 μM AF [22]. However, in the AF‐sensitive CMT cell line CHMp, ubiquitinated protein accumulation at 10 nM Bz did not significantly differ from the control group (Figure S3). This suggests that CMT cells may exhibit higher resistance to proteasome inhibition than human breast cancer cells, requiring higher Bz concentrations to achieve effective proteasome inhibition.

ER stress occurs when the ER's protein‐folding capacity is overwhelmed, leading to an accumulation of misfolded or unfolded proteins [32, 33, 34]. This activates the UPR and ISR, which restores proteostasis or induces cell death under prolonged stress [32, 33, 34, 35, 36]. The UPR plays a central role in managing ER stress, with IRE1α as a key mediator [32, 33, 34]. IRE1α drives the expression of genes that alleviate the protein‐folding burden [32, 34]. Complementing the UPR, the ISR integrates various stress signals, such as ER stress, nutrient deprivation, and oxidative stress [35, 36]. ISR activation promotes the translation of stress‐responsive transcripts like ATF4 [35, 36]. ATF4 regulates genes involved in programmed cell death, with persistent ISR activation often marking the transition from adaptation to cell death [34, 35, 36]. In AF‐sensitive CMT cells, IRE1α expression initially increased, indicating early UPR activation aimed at restoring ER proteostasis. With increasing concentrations of AF treatment, IRE1α expression decreased while ATF4 expression rose, reflecting a shift from UPR‐mediated adaptation to ISR activation. This transition indicates that the stress exceeded the tolerance of sensitive cells, leading them to initiate programmed cell death. Conversely, in AF‐resistant CMT cells treated with AF + Bz, IRE1α expression was increased, whereas ATF4 expression remained unchanged. This suggests that the full transition from UPR to ISR has not yet occurred. Interestingly, paraptosis‐like cell death was observed despite the absence of ATF4 upregulation, highlighting that this process can occur independently of ATF4 upregulation. In contrast, ATF4 upregulation has been identified as a critical regulator of proteotoxic stress and paraptosis in human breast cancer cells [22]. These differences may reflect higher baseline proteasome activity in CMT cells and corresponding variations in their ER stress response pathways.

Paraptosis is a distinct form of programmed cell death characterised by cytoplasmic vacuolation originating from the ER and/or mitochondria [19]. In our study, AF‐resistant cells treated with a combination of AF and Bz exhibited extensive cytoplasmic vacuoles primarily originating from the ER, a hallmark feature of paraptosis. Additionally, pretreatment with CHX, known as a paraptosis inhibitor, effectively reversed the vacuolation and partially restored cell viability. These findings suggest that vacuole formation is not a resistance mechanism but rather contributes to reduced viability, supporting the induction of a paraptosis‐like mechanism by AF + Bz. Considerable evidence connects ER stress and proteasome inhibition to the induction of paraptosis [20, 37, 38, 39, 40]. Since Bz alone can induce ER stress, its combination with AF amplifies this stress, leading to paraptosis‐like cell death. In this study, AF‐resistant cell lines treated with AF + Bz demonstrated a marked accumulation of ubiquitinated proteins and pronounced elevations in ER stress markers. These findings indicate significant disruption of proteostasis and the resulting overburden of misfolded proteins in the ER. The accumulation of misfolded proteins in the ER can exacerbate osmotic imbalances, potentially driving water influx and the formation of vacuoles within the ER [21, 41, 42]. This mechanism may explain our observation of extensive cytoplasmic vacuolation in AF‐resistant cells treated with AF + Bz. In contrast, AF‐sensitive cell lines did not display cytoplasmic vacuolation even when treated with lethal AF doses inhibiting proteasome activity (Figures S4 and S5). This suggests that AF‐sensitive cells may undergo apoptosis before vacuolation develops, highlighting potential differences in cell death pathways between AF‐sensitive and AF‐resistant cell lines. Further investigation into these two distinct cell death pathways and differing sensitivity to AF across CMT cell lines could provide valuable insights for the development of targeted therapeutic strategies.

Several studies have investigated the pharmacokinetics and safety profile of AF in humans and animals, supporting its potential application in CMT. In dogs, a single oral dose of 0.1 mg/kg AF produced a blood gold (Au) maximum concentration of approximately 0.2 μM (40 ng Au/mL) [43]. Since each AF molecule contains one gold atom, the molar concentrations of AF and Au are equivalent. Oral AF doses up to 6 mg/kg/day were well tolerated in dogs without excessive emesis, and prenatal development studies in rats established a no‐observed‐adverse‐effect level (NOAEL) of 5 mg/kg/day for maternal and fetal toxicity [44]. The therapeutic strategies proposed in this study include using up to 2 μM AF as monotherapy in AF‐sensitive cells and combining 2 μM AF with Bz to treat resistant cells. Proportional extrapolation suggests that achieving a 2 μM maximum plasma concentration (Cmax) in dogs would require single oral doses of approximately 1 mg/kg. This dose is higher than the current oral clinical dose in dogs but remains well below the NOAEL. Furthermore, prolonged administration could potentially achieve similar levels even with lower doses. For instance, in human clinical settings, AF is typically administered orally at a dose of 6 mg/day [45, 46] and a simulation study indicated that this regimen over 14 days achieves a steady‐state blood gold concentration of 0.42 μg/mL (2.03 μM) [47]. Additionally, FDA‐approved trials (NCT01419691) for CLL allowed daily doses up to 21 mg, with patients tolerating up to 12 mg/day for 28 days [48]. With regard to tolerance in dogs, AF demonstrated a favourable safety profile when administered orally at a dosage of 9 mg (for dogs ≥ 15 kg) or 6 mg (for dogs < 15 kg) every 3 days in a study involving dogs with osteosarcoma, where treatment was continued until relapse or death [28]. Unlike conventional chemotherapy, which is often accompanied by severe side effects, AF showed no significant abnormalities in blood screen metrics, and no unexpected symptoms or behavioural changes were reported by the owners [28]. These findings suggest that achieving a blood Cmax near 2 μM of AF in dogs is feasible, and AF demonstrates advantages in safety and tolerability compared to conventional chemotherapy, positioning it as a promising treatment option for CMT.

In conclusion, our study demonstrates that AF induces ER stress‐mediated apoptosis in AF‐sensitive CMT cells, and its combination with Bz induces paraptosis‐like cell death in AF‐resistant CMT cells. These findings suggest that AF, alone or in combination with Bz, represent a promising therapeutic strategy for malignant CMT, highlighting paraptosis‐like cell death as a potential alternative approach to target canine tumour cells.

Author Contributions

Yoon‐Ho Suh conceptualised and designed the study, developed the methodology, performed the experiments, analysed the data, wrote the original draft, and edited the manuscript. Se‐Hoon Kim contributed to the study design, methodology development, data analysis, and manuscript revision. Ki‐Hoon Song contributed to the methodology development, data analysis, and manuscript revision. Jun‐Yeol Choi contributed to the methodology development and data analysis. Min‐Ok Ryu and Robert. B. Rebhun reviewed the manuscript. Kyoung‐Won Seo conceptualised and designed the study, supervised the overall research, contributed to data interpretation, critically revised the manuscript, and was responsible for the final approval of the version to be published. All authors read and approved the final version of the manuscript.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1. Supporting Information.

VCO-23-377-s001.docx (4.8MB, docx)

Acknowledgements

This study was partially supported by the Research Institute for Veterinary Science, Seoul National University. The authors thank the Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, for providing the canine mammary tumour cell lines CIPp, CIPm, CHMp, and CHMm.

Suh Y.‐H., Kim S.‐H., Song K.‐H., et al., “Auranofin Induces ER Stress‐Mediated Apoptosis, and Its Combination With Bortezomib Elicits Paraptosis‐Like Cell Death in Malignant Canine Mammary Tumour Cells,” Veterinary and Comparative Oncology 23, no. 3 (2025): 377–387, 10.1111/vco.13062.

Funding: This work was supported by the Research Institute for Veterinary Science, Seoul National University.

The views and opinions expressed in this article are solely those of the authors and do not reflect the official position of the institution or funder. This work was conducted independently and should not be considered as representative of the policies or positions of any affiliated organisations.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1. Supporting Information.

VCO-23-377-s001.docx (4.8MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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