Targeting mitochondrial function to inhibit glycolysis and oxidative phosphorylation (OXPHOS) for therapeutic intervention in triple-negative breast cancer line (MDA-MB 231) utilising Cape fynbos

• Five Cape fynbos plants show cytotoxicity against MDA-MB 231 cells, with IC50 values < 30 µg/mL. • Metabolite profiling identified fatty acyls, phenols, flavonoids, coumarins and organooxygen compounds. • Plant extracts activate apoptosis pathways, confirmed by caspase activities, similar to cisplatin. • Elevated glycolytic and mitochondrial respiration activities were observed in the extracts except for E. pulchella. Triple-negative breast cancer (TNBC) presents a significant clinical hurdle due to its aggressive nature and limited therapeutic options. Despite the efficacy of chemotherapy, such as taxanes like paclitaxel, the emergence of drug resistance necessitates the exploration of alternative treatments. This study investigates the potential of five Cape fynbos plant species ( Erica plukenetii L., Erica imbricata L., Erica sessiliflora L.f., Indigofera hamulosa Schltr., and Erica pulchella Houtt) as novel therapeutic agents for TNBC. Hexane and methanolic extractions of plant materials were conducted, followed by mass spectrometry-based metabolite profiling to identify phytochemical constituents. Cytotoxicity against MDA-MB 231 TNBC cells was assessed alongside flow cytometry to confirm the mode of cell death and multiplex activity assays for caspase activity. Glycolysis and mitochondrial function were evaluated using the Agilent Seahorse XFp Analyser and Oroboros O2K, respectively. Metabolite profiling revealed the presence of fatty acyls, phenols, flavonoids, coumarins and organooxygen derivatives. The extracts exhibited significant cytotoxicity, with IC 50 values of <30 µg/mL for I. hamulosa, E. plukinetii and E. sessiliflora . Apoptosis induction ranged from 13.09 % to 15.82 %, similar to the efficacy of the reference drug cisplatin (20.25 %). Caspase 3, 8, and 9 activities confirmed the activation of the apoptosis pathways by both cisplatin and plant extracts. Elevated glycolytic and mitochondrial respiration activities were observed in the extracts compared to controls, except for E. pulchella . These findings highlight the potential of Cape fynbos plants as promising therapeutic candidates for TNBC, offering a pathway for the development of targeted treatments with minimal adverse effects .