(O16) Auranofin Induces Apoptosis and Contributes to Paraptosis-Like Cell Death in Malignant Canine Mammary Tumor Cells

Background: Canine mammary tumors (CMT) are common in female dogs, often exhibiting high malignancy and limited responses. Auranofin (AF), an FDA-approved drug for human rheumatoid arthritis that is also used in dogs to treat idiopathic polyarthritis, has demonstrated anticancer effects in various cancers.

Objectives: To evaluate the anticancer effects and mechanisms of AF in five malignant CMT cell lines, focusing on its ability to induce distinct forms of cell death.

Methods: Cells were treated with AF under various conditions, and subsequent assessments included cell viability, clonogenicity, thioredoxin reductase (TrxR) activity, and protein expression. Additionally, live-cell staining was performed to investigate the origin of cytoplasmic vacuoles.

Results: AF inhibited TrxR activity, cell proliferation, and colony formation across malignant CMT cell lines. In AF-sensitive cell lines, 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. Therefore, we combined sublethal doses of AF (~2 µM) with the proteasome inhibitor Bortezomib (Bz) in AF-resistant cell lines. The combination exhibited synergistic cytotoxicity and induced extensive cytoplasmic vacuolation originating from the ER. Cycloheximide pretreatment inhibited both vacuolation and AF + Bz-induced cell death, indicating features of paraptosis. This cell death was related to enhanced ER stress from AF-induced TrxR inhibition and Bz-induced proteasome inhibition.
Conclusions and clinical importance: We propose AF alone or with Bz as a therapeutic strategy for malignant CMT. This study highlights AF’s potential to induce ER stress-mediated apoptosis and paraptosis-like cell death, expanding options for canine cancer treatment.