High-throughput, Label-Free Quantitative Proteomic Studies of the Anticancer Effects of Electrical Pulses with...

Triple negative breast cancer (TNBC) represents 15-20% of the over one million new breast cancer cases occurring each year. TNBC is an aggressive cancer phenotype, with low 5-year survival rates, high 3-year recurrence rates, and increased risk of metastasis. A lack of three commonly exploited hormone receptors renders TNBC resistant to endocrine therapies and lends to its critical absence of viable therapeutic targets. This necessitates the development of alternate and effective novel therapeutic strategies for TNBC. Towards this, our current work seeks to develop the technique of Electrical pulse (EP)-mediated Turmeric silver nanoparticles (TurNP) therapy, known as Electrochemotherapy (ECT), to effectively target TNBC cells. This technique involves the efficient delivery of natural bioactive molecules with anti-cancer effects via a biophysical means. In these experiments, the bioactive molecules are turmeric, a dried rhizome of Curcuma longa that has been used for centuries, both as a dietary supplement and as a medicine in Ayurveda (science of life) in the Indian subcontinent and in traditional Chinese medicine. Our results reveal the combined effect of TurNP + EP treatment in reducing MDA-MB-231 cell viability to as low as 9% at 12 h. Showing biological selectivity, this combination treatment has a substantially lower effect on non-tumorigenic mammary epithelial MCF10A cells (67% viability). To gain mechanistic insights into the actions of TurNP-based ECT treatment, we performed high-throughput, label-free quantitative proteomics studies. Proteomics results indicate that TurNP + EP treatment significantly influenced expression of a diverse list of proteins, including receptors, transcription factors, structural proteins, kinases, and metabolic enzymes. This include the downregulation of 25 proteins in PI3K-Akt signaling pathway (such as GRB2, EGFR, EPHA2, GNB1, GNB2, 14-3-3 family, and Integrin family proteins), and 12 proteins (AKR1A1, ALDOA, ALDOC, PGK1, PGM1, PGAM1, ENO1, ENO2, GAPDH, TPI1, LDHA, and LDHB) in the glycolytic pathway with concomitant reduction in metabolite levels (glucose uptake, and intracellular- lactate, glutamine, and glutamate). Compared to TurNP alone, TurNP + EP treatment upregulated 66 endoplasmic reticulum and 193 mitochondrial proteins, enhancing several processes and pathways, including Pyruvate Metabolism, Tricarboxylic acid (TCA) cycle, and Oxidative Phosphorylation (OXPHOS), which redirected the TNBC metabolism to mitochondria. This switch in the metabolism caused excessive production of H₂O₂ reactive oxygen species (ROS) to inflict cell death in MDA-MB-231 cells, demonstrating the potency of this treatment.