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

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.