Antitumor Effects of Esculetin, a Natural Coumarin Derivative, against Canine Mammary Gland Tumor Cells by Inducing Cell Cycle Arrest and Apoptosis

SIMPLE SUMMARY: Canine mammary gland tumors (CMTs) are the most common type of tumor in female dogs, and approximately 50% of them are diagnosed as malignant. Currently, several chemotherapeutic agents have been applied to treat those CMTs that are difficult to remove by surgery, due to metastasis. However, it is necessary to discover more safe and specific drugs for the treatment of CMTs. This research investigates the anticancer effects of esculetin, a natural coumarin derivative, and its underlying mechanisms on CMT cell lines, CMT-U27 and CF41.mg. Esculetin remarkably inhibited the viability and migration of both cell lines. Esculetin treatment activated the protein expression of caspase 3, a typical marker of apoptosis, leading to apoptotic cell death. Furthermore, esculetin promoted cell cycle arrest in both cell lines. Interestingly, esculetin has potently reduced the protein expression of CDK4 and cyclin D1, regulators of G1/S transition, in both cell lines; however, cell cycle arrest was caused at a different phase in both cell lines, i.e., at the G0/G1 phase in CMT-U27 cells and the S phase in CF41.mg cells. These results demonstrate the anticancer effect of esculetin and lay a theoretical foundation for in vivo experiments and clinical trials. ABSTRACT: Mammary gland tumors are the most common neoplasms in female dogs, of which 50% are malignant. Esculetin, a coumarin derivative, reportedly induces death in different types of cancer cells. In this study, we explore the anticancer effects of esculetin against CMT-U27 and CF41.mg canine mammary gland tumor cells. Esculetin significantly inhibited the viability and migration of both CMT-U27 and CF41.mg cells in a dose- and time-dependent manner. Flow cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed increased numbers of annexin-V-positive cells and DNA fragmentation. Furthermore, a cell cycle analysis demonstrated that esculetin blocked the cell progression at the G0/G1 phase and the S phase in CMT-U27 and CF41.mg cells. These results were supported by a Western blot analysis, which revealed upregulated protein expression of cleaved caspase-3, a marker of apoptosis, and downregulated cyclin-dependent kinase 4 and cyclin D1 protein, the cell cycle regulators. In conclusion, this novel study proves that esculetin exerts in vitro antitumor effects by inducing apoptosis and cell cycle arrest in canine mammary gland tumors.