Rosmarinic Acid Enhances Doxorubicin Activity in MDA-MB-231 Cells: Associations with Intracellular Oxidative Stress,...

Background/Objectives: Rosmarinic acid (RA) is a naturally occurring polyphenolic compound with promising anticancer activity; however, its potential to enhance the therapeutic efficacy of doxorubicin (DOX) against triple-negative breast cancer (TNBC) has not been comprehensively characterised. This study investigated the pharmacological interaction and associated cellular responses of RA combined with DOX in MDA-MB-231 breast cancer cells, while including HaCaT human keratinocytes as a non-malignant, non-mammary reference model for comparative cytotoxicity assessment. Methods: Cell viability was assessed using the MTT assay, and pharmacological interactions were evaluated using Chou-Talalay combination index (CI) and dose reduction index (DRI) analyses. Intracellular oxidative activity was evaluated using DCFH-DA fluorescence analyses together with N-acetyl-L-cysteine (NAC) modulation experiments. Apoptosis, cell-cycle distribution, mitochondrial membrane potential (JC-1), Caspase-9 immunocytochemistry, nuclear morphology (NucBlue staining), apoptosis- and proliferation-related gene expression (RT-qPCR; BAX, BCL2, CASP3, CASP9, TP53, CDKN1A, MKI67, and PCNA), and bioinformatic pathway analyses were performed to characterise cellular and molecular responses associated with the combined treatment. Results: After 48 h, RA exhibited IC₅₀ values of 188.4 ± 4.2 µM in MDA-MB-231 cells and 146.6 ± 6.8 µM in HaCaT cells, while DOX showed IC₅₀ values of 1.2 ± 0.08 µM and 2.6 ± 0.15 µM, respectively. The Chou-Talalay analysis demonstrated synergistic interactions in MDA-MB-231 cells, with CI values of 0.86, 0.72, and 0.64 at the effect levels of IC₂₅, IC₅₀, and IC₇₅, respectively; the model-derived DOX DRI value at Fa = 0.50 was 1.8. Combination treatment markedly increased intracellular DCFH-DA fluorescence, whereas NAC pretreatment attenuated this signal and partially restored cell viability, supporting a contributory role of intracellular oxidative stress in treatment-associated cytotoxicity. Increased intracellular oxidative activity coincided with mitochondrial membrane depolarisation, increased apoptotic cell death, accumulation of cells in the sub-G₁ phase, enhanced Caspase-9 immunoreactivity, and pronounced apoptotic nuclear alterations. RT-qPCR analysis demonstrated significant upregulation of BAX, CASP3, CASP9, and TP53, together with downregulation of BCL2, MKI67, and PCNA, resulting in a marked reduction in the BCL2/BAX mRNA expression ratio. A modest but non-significant increase in CDKN1A expression was also observed. Bioinformatic analyses further identified predicted associations with mitochondrial apoptosis and p53-associated signalling pathways. Conclusions: The RA + DOX combination showed synergistic cytotoxic activity in MDA-MB-231 cells, accompanied by increased intracellular oxidative activity, mitochondrial membrane depolarisation, apoptosis, cell-cycle perturbation, and changes in proliferation-related gene expression. These findings indicate associated cellular responses but do not establish a ROS-dependent mitochondrial apoptotic mechanism. The findings provide an exploratory in vitro basis for further investigation of the RA + DOX combination in additional TNBC models, non-malignant mammary epithelial cells, and appropriate in vivo systems.