Eugenoplatin (a phyto-chemo drug conjugate) targets cancer stem cells through inhibition of the β-catenin signaling in triple-negative breast cancer cells

Breast cancer is the most prevalent and leading cause of cancer-related mortality among women worldwide. The triple-negative subtype, the most aggressive form of the disease, is associated with the poorest prognosis. In order to improve the treatment of these patients, we have synthesized a phyto-chemo drug conjugate composed of cisplatin and eugenol, two molecules with synergistic anti-cancer effects. Eugenoplatin was synthesized and characterized by HPLC, LC/MS, and 1H-NMR, while its physicochemical properties were predicted by in silico analysis using the Biovia Discovery Studio. The cytotoxic effects of eugenoplatin were first tested in vitro using the WST1 and the flow cytometry techniques. Cell proliferation, migration, and invasion abilities were assessed using the xCELLigence Real-Time Cell Analysis. The changes in gene expression were determined by proteomics analysis (LC/MS), immunoblotting, immunofluorescence, and quantitative RT-PCR. The effect of the drug on breast cancer stem cells was determined by the tumorsphere formation assay, and cell sorting was utilized to isolate a specific breast cancer sub-population of cells. Furthermore, orthotopic tumor xenografts were used to test the potential therapeutic value of eugenoplatin in vivo. The novel DNA-damaging molecule (eugenoplatin) has shown higher cytotoxicity through induction of apoptosis, and strong inhibition of cell growth via cell cycle delay at G2/M phase in different cell lines. In addition, eugenoplatin targeted cancer stem cells through inhibition of the β-catenin signaling pathway. These effects were confirmed in humanized orthotopic tumor xenografts, wherein the eugenoplatin-dependent tumor growth inhibition was more than 10-fold stronger than the cisplatin repressive effect, with a potent inhibitory effect on cancer stem cell biomarkers. This was associated with the suppression of various cancer-related signaling pathways, including STAT3/NF-κB, AKT/mTOR, and ERK. Furthermore, eugenoplatin down-regulated the pro-angiogenic factors VEGF-A, IL-6, and IL-8, and repressed angiogenesis both in vitro and in tumor xenografts. In silico analysis has suggested good permeability, good absorption, and higher bioavailability for eugenoplatin, which is also unlikely to be mutagenic, carcinogenic, or hepatotoxic. These results indicate that eugenoplatin, a novel DNA-damaging agent that can also target cancer stem cells, could be of great therapeutic value for TNBC patients.