Proteasome Inhibitor-Induced Cytotoxic Mechanisms in Canine Pulmonary Adenocarcinoma Cell Lines.

Canine pulmonary adenocarcinoma (cPAC) lacks well-validated systemic treatment options, motivating preclinical identification of potential therapeutic candidates. To explore potential therapeutic candidates, we first performed a screening of an FDA-approved drug library using a cPAC cell line, which identified bortezomib-a proteasome inhibitor-as a potential agent. Guided by this result, we focused subsequent investigations on proteasome blockade and evaluated the antitumor activity and mechanisms of bortezomib together with a second-generation proteasome inhibitor, carfilzomib-alone and in combination with standard cytotoxics-in three cPAC cell lines (CLAC, HDC, and LuBi). Both agents reduced cell proliferation and cell viability in a dose-dependent manner, with proteasome target engagement (ubiquitinated protein accumulation), G2/M arrest, sub-G1 increase, and caspase-3 cleavage. Pan- and selective caspase inhibition partially rescued viability, suggesting contributions from both intrinsic and extrinsic apoptosis, together with caspase-independent mechanisms. We also observed activation of the JNK-c-Jun pathway and increased Bim expression, indicating the induction of cellular stress responses and apoptosis. Drug-interaction analysis showed reproducible synergy between carfilzomib and carboplatin in HDC and LuBi, whereas combinations with vinorelbine were mainly additive or antagonistic effects. Overall, proteasome inhibitors exert multifaceted cytotoxic effects in cPAC through proteotoxic stress, cell-cycle arrest, and caspase-associated apoptosis. In particular, the combination of carfilzomib and carboplatin may represent a promising therapeutic strategy. Further in vivo and clinical investigations are warranted to evaluate therapeutic efficacy, safety, and pharmacokinetics of proteasome inhibitors in cPAC.