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.

Methyl gallate is a phenolic compound mainly found in medicinal plants. It has been reported to its anticancer activity in various tumors. In this study, we aimed to demonstrate the antitumor effect of methyl gallate in the melanoma mouse model and B16F10 cells. Our results showed that methyl gallate decreased cell viability and induced apoptosis by increasing the expression of cleaved caspase3 in B16F10 cells and prevented cell migration and tube formation in human umbilical vein endothelial cells. In B16F10 cell-inoculated mice, methyl gallate not only decreased tumor volume by 30% but also significantly reduced tumor vessel density and pericyte coverage. Moreover, methyl gallate diminished by close to 50% the expression of cytokeratin and LYVE-1 in mouse right inguinal lymph nodes, indicating that methyl gallate could suppress metastasis. In conclusion, this study suggests that methyl gallate inhibits tumor development by inducing apoptosis and blocking tumor angiogenesis and metastasis and might be considered a therapeutic agent for melanoma.