Synergistic Effects of Epirubicin‐Vorinostat‐Pimozide Drug Cocktail on Proliferation, Stemness, Invasiveness, and Fatty Acid Metabolism in Breast Cancer Cells

Chemotherapeutic treatments for breast cancer are often associated with severe toxicity due to the requirement of high concentrations of the drugs for efficacy. The combination of chemotherapy drugs along with repurposed drugs offers a promising strategy to enhance efficacy while reducing toxicity. However, the effectiveness of such combinations is likely to be hindered by improper metabolism of the drugs due to the sharing of the same metabolizing enzymes. In this study, we explored a novel approach to enhance the efficacy of Pimozide (repurposed drug) by combining it with chemotherapeutic drugs that utilize different metabolizing enzymes than Pimozide, thereby reducing metabolic load and toxicity. The Epirubicin-SAHA(Vorinostat)-Pimozide (ESP) combination emerged as highly synergistic, reducing the IC₅₀ of Pimozide from 16.54 to 0.57 μM in MCF-7 cells and from 17.5 to 3.35 μM in MDA-MB-231 cells, representing a significant enhancement in efficacy. Mechanistic studies revealed increased intracellular reactive oxygen species (ROS) generation and activation of the intrinsic apoptosis pathway, as indicated by a 10-fold increase in the cleaved PARP levels. In MDA-MB-231 cells, there was also a 2-fold increase in p53 and a 10-fold increase in p21 expression, with a concomitant reduction in AKT signaling. Furthermore, the ESP combination reduced cancer stemness, invasiveness, fatty acid uptake, and lipid droplet accumulation, pointing to its broad impact on cancer cell survival and metabolism. These findings suggest that the ESP combination holds promise as an effective therapeutic strategy for breast cancer, with reduced toxicity and enhanced efficacy.