Effects of Rosmarinic Acid and Doxorubicin Combination in Breast Cancer Cells

Rosmarinic acid (RA), a naturally occurring polyphenolic compound, has demonstrated promising anticancer activity; however, its combinatorial potential with conventional chemotherapeutic agents remains incompletely characterized. This study investigated the cytotoxic, pro-apoptotic, oxidative stress-associated, and cytokine-associated effects of RA alone and in combination with doxorubicin (DOX) in 4T1 murine breast cancer cells and HaCaT human keratinocyte cells as a non-cancerous control model. Cellular viability, apoptosis, cell cycle progression, oxidative stress, mitochondrial function, cytokine responses, and apoptosis-associated molecular alterations were evaluated using complementary cellular and molecular approaches. In addition, three-dimensional (3D) tumor spheroid experiments were performed to assess treatment responses under physiologically relevant tumor-like conditions. Results demonstrated that RA synergistically enhanced DOX-induced cytotoxicity in 4T1 cells while exhibiting comparatively lower toxicity toward HaCaT cells. Combination treatment significantly increased apoptotic cell death, mitochondrial depolarization, intracellular reactive oxygen species (ROS) accumulation, apoptotic DNA fragmentation, and G₂/M-phase accumulation. N-acetylcysteine (NAC)-mediated rescue experiments partially reversed ROS elevation and treatment-associated cytotoxicity in both monolayer and 3D spheroid models. Furthermore, the RA+DOX combination markedly disrupted spheroid integrity and reduced spheroid viability compared with monotherapies. Collectively, these findings indicate that RA enhances the anticancer activity of DOX and support further investigation of this combination strategy in breast cancer models.