Simultaneous proteasome and autophagy inhibition synergistically enhances cytotoxicity of doxorubicin in breast cancer cells

Ubiquitin-proteasome system (UPS) and autophagy are interconnected proteolysis pathways implicated in doxorubicin resistance of breast cancer cells. Following anticancer treatments, autophagy either plays a cytoprotective role or augments treatment-induced cytotoxicity. However, the role of autophagy in breast cancer cells cotreated with doxorubicin and ixazomib remains unclear. The expression of autophagy proteins (LC3A/B and Beclin-1) and UPS protein (ubiquitin) in MDA-MB-231 and MCF-7 cells following doxorubicin, ixazomib, and/or hydroxychloroquine were determined by western blot. The combinatorial effects and combination index (CI) of triple-combination were determined by cell viability assay and CompuSyn software, respectively. Doxorubicin and ixazomib cotreatment increased Beclin-1 (3.8- and 3.5-fold) and LC3-II expression (13.5- and 1.9-fold) in MDA-MB-231 and MCF-7 cells, respectively. Adding lysosomal inhibitor hydroxychloroquine to doxorubicin and ixazomib further increased LC3-II expression to 45.0- and 16.5-fold in MDA-MB-231 and MCF-7 cells, respectively, confirming autophagy induction. The triple-combination synergistically inhibited cell growth, achieving CI 0.672 and 0.157 in MDA-MB-231 and MCF-7 cells, respectively. The triple-combination also induced ubiquitinated proteins accumulation (2.5-fold and 3.0-fold) in MDA-MB-231 and MCF-7 cells, respectively. These results suggest that the autophagy induced by doxorubicin and ixazomib cotreatment serves cytoprotective role in breast cancer cells. Simultaneous UPS and autophagy inhibition synergistically enhanced doxorubicin-mediated cytotoxicity.