Investigations of potential therapeutic targets from high-throughput, label-free, quantitative proteomic studies in MDA-MB-468 cells treated with metformin and electrical pulses.

High-throughput, label-free quantitative proteomic analyses, along with cell viability and reactive oxygen species (ROS) studies, were performed on MDA-MB-468 human triple-negative breast cancer (TNBC) cells, to gain mechanistic insights into therapeutic actions. TNBC was chosen as it is the most lethal subtype of breast cancer, highly aggressive, prone to recurrence and metastasis, with the highest prevalence in black women. The absence of specific biomarkers limits targeted therapies. This unmet need was studied using proteomics, after treating the cells with electrical pulses (EP) combined with metformin (met). With the EP + met treatment (1000 V/cm, 1 mM), cell viability dropped to 25.6 % after 24 h and ROS increased to 179 %, compared to control at 100 %. Proteomics revealed 125 upregulated and 37 downregulated proteins in EP + met, compared to met alone, involving enzymes, proliferation markers, and kinases. Key gene changes included upregulation of ALAD, MKI67, and LRCH4, and downregulation of EIF1AX, NSUN5, and GNS. LRCH4 and GNS are suggested to be potential novel therapeutic targets, as LRCH4 upregulation links to inhibition of the mTOR/PI3K/Akt pathway, reducing proliferation, while GNS downregulation suppresses tumor growth and metastasis. Overall, proteomics-based preliminary findings suggest that EP + met modulate TNBC pathways, identifying potential biomarkers and providing a foundation for future validation.