Synergy between sublethal doses of shikonin and metformin fully inhibits breast cancer cell migration and reverses epithelial-mesenchymal transition

Background: Shikonin is a natural multipotent anti-tumorigenic compound. We investigated potential synergy between shikonin and anti-diabetic metformin against tumorigenic properties of breast cancer cell line MCF-7. Methods and results: The IC of shikonin and metformin was determined after a single treatment of two cell lines MCF-7 and MDA-MB-231. We then measured optimal doses of each drug, used in combination, in MCF-7 cells. These sub-IC doses were co-applied for all subsequent combined treatments to evaluate their synergistic effects on MCF-7 tumorigenic properties. Next, we examined expression levels of the genes crucial for apoptosis, cell growth, and EMT using RT-PCR or real-time PCR and monitored CD44/CD24 ratios using flow cytometry. Binding energies between shikonin and growth molecules were measured by in silico simulation. Shikonin caused significantly reduced cell survival that was accelerated by the synergizing presence of metformin. Drug combination induced apoptosis and ROS levels while fully blocking cell migration and reverting EMT. RT-PCR showed strong suppression of BCL-2 but induction of BAX and PTEN. Prolonged shikonin treatment caused a total loss of the nuclear membrane, whereas metformin prevented this damage while promoting apoptotic morphologies. Our real-time PCR detected reduced levels of EMT genes but increases in the anti-EMT gene CDH1. Combined treatment also reduced CD44/CD24 ratios in favor of chemosensitivity. Binding energies strongly favored shikonin interactions with growth-signaling molecules. Conclusions: Shikonin and metformin synergize in inhibiting the tumorigenic activities of MCF-7 cells including their proliferation, invasiveness, and EMT with a potential to inhibit multidrug resistance.