Background/Objectives: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited targeted therapeutic options. Although the anticancer and pro-apoptotic properties of boswellic acids have previously been reported, the interaction profile of chemically defined β-boswellic acid (BA) with doxorubicin (DOX) remains insufficiently characterised in the murine 4T1 TNBC-like model. This study quantitatively evaluated BA–DOX drug interactions using different reference models and characterised the cytotoxic and apoptosis-associated cellular phenotype accompanying combined exposure.
Methods: Cytotoxicity was assessed using the MTT assay and BA–DOX interactions were evaluated using the Chou–Talalay combination index (CI), highest single-agent (HSA) and Bliss independence models. Apoptosis and cell-cycle distribution were analysed by flow cytometry and mitochondrial membrane potential was assessed by JC-1 staining. Caspase-3/7 activity, RT-qPCR, live/dead Calcein-AM/PI staining, 4′,6-Diamidino-2-phenylindole (DAPI) nuclear staining, and cytokine measurements were also performed. Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), and STRING-based protein–protein interaction (PPI) analyses were used to explore putative molecular pathways associated with experimental findings.
Results: The 48 h selectivity index of BA was 1.28, indicating only modest differential cytotoxicity between 4T1 cells and HaCaT keratinocytes under the experimental conditions rather than definitive cancer-cell selectivity. Drug-interaction analyses revealed concentration- and model-dependent effects, with the Chou–Talalay analysis indicating synergism in selected intermediate and higher concentration pairs. Under the selected phenotypic-characterisation condition, BA + DOX produced a greater apoptotic response than either single treatment, accompanied by increased G2/M and Sub-G1 fractions, mitochondrial membrane depolarisation, and increased caspase-3/7 activity. This treatment condition was not included in the drug-interaction analysis and was therefore not interpreted as a pharmacologically validated synergistic combination. RT-qPCR demonstrated increased mRNA expression of
Bax,
Casp3, and
Casp9, decreased mRNA expression of
Bcl2, and a marked increase in the
Bax/
Bcl2 mRNA ratio. Calcein-AM/PI and DAPI analyses further demonstrated increased cell death and apoptotic nuclear alterations. The measured concentrations of TNF-α and IL-6 in culture supernatants were lower after BA + DOX treatment, whereas IL-10 remained unchanged; however, these cytokine measurements were not normalised to viable cell number and therefore require cautious interpretation. Exploratory bioinformatic analyses identified predicted associations with apoptosis-, mitochondrial-, and cell-cycle-related processes and pathways; however, these database-derived findings were considered hypothesis-generating and not evidence of BA-dependent target engagement or pathway activation.
Conclusions: Combined BA and DOX exposure produced greater cytotoxic and apoptosis-associated responses than either single treatment in 4T1 cells, whereas formal drug-interaction classifications varied according to concentration and analytical model. The accompanying changes in mitochondrial membrane potential, caspase-3/7 activity, and apoptosis-related gene expression describe a treatment-associated cellular phenotype but do not identify a direct molecular target of BA or establish a causal molecular mechanism. The findings also do not demonstrate TNBC-specific selectivity. Further studies using additional breast cancer and tissue-matched non-malignant models, together with direct target-engagement and functional pathway-validation approaches, are required.
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