Methyl divanillate enhances doxorubicin efficacy in TNBC cell lines through redox imbalance and apoptotic reprogramming.

Triple-negative breast cancer (TNBC) represents one of the most aggressive breast cancer subtypes, lacking effective targeted therapies and relying primarily on chemotherapy. In this study, we investigated the cytotoxic and molecular effects of doxorubicin (DOX) and methyl divanillate (DMV), alone or in combination, in three-dimensional spheroid models derived from MDA-MB-231 (claudin-low) and HCC70 (basal-like) TNBC cell lines. Combined DOX/DMV treatment significantly reduced spheroid viability and was associated with increased reactive oxygen species (ROS) accumulation and thiol depletion, thereby indicating redox imbalance and cytoskeletal disruption. Immunofluorescence further confirmed actin filament disorganization and nuclear instability under combined therapy. Integrated qPCR and proteomic analyses revealed lineage-dependent modulation of apoptotic signaling in MDA-MB-231 spheroids, DOX/DMV broadly suppressed apoptosis-associated proteins, whereas in HCC70 spheroids, the combination upregulated key intrinsic apoptotic regulators, including caspases and mitochondrial stress-related proteins. Furthermore, gene expression analysis, gelatin zymography, and proteomic profiling demonstrated that DOX and DMV modulated metalloproteinase (MMP) activity and cytoskeletal remodeling, with combination therapy promoting MMP9 suppression and widespread downregulation of actin cytoskeleton regulators in MDA-MB-231 spheroids, while inducing cytoskeletal reinforcement-associated profiles in HCC70 spheroids. Transgelin-2 expression also exhibited divergent patterns between the cell lines at both transcript and protein levels. Collectively, our findings indicate that DOX/DMV combination therapy exerts synergistic interaction, as determined by Combination Index analysis (CI = 0.3 for MDA-MB-231 spheroids and CI = 0.8 for HCC70 spheroids), resulting in enhanced cytotoxicity associated with oxidative stress, apoptotic pathway modulation, and cytoskeletal remodeling in a subtype-dependent manner. These results underscore the potential of DMV as a redox-modulating adjuvant to enhance chemotherapy efficacy in TNBC, while highlighting the importance of tumor subtype context in determining therapeutic responses.