4-methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells.

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is synthesized by HA synthase (HAS) 1, HAS2 and HAS3 and is intricately involved in cell growth and metastasis. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has been reported to exhibit anticancer properties in various types of malignant tumors. However, the underlying mechanisms at the molecular and cellular levels remain unclear. In this study, to establish an animal model for studying the function of HA in human breast cancer, we investigated the antitumor effects of 4-MU using canine mammary tumor (CF33) cells. First, we investigated the effects of 4-MU on HA production in CF33 cells. Quantitative analysis of HA in culture media showed that 4-MU inhibited HA synthesis, accompanied by downregulation of HAS2 mRNA levels, in a dose-dependent manner at 24-72 h. Additionally, we observed a 4-MU-mediated decrease in the extent of the cell-associated HA matrix. We examined the effect of 4-MU on cell growth and apoptosis in CF33 cells. 4-MU markedly inhibited cell proliferation and induced apoptosis in CF33 cells. In particular, our experiments showed that the mechanism of 4-MU-induced apoptosis in CF33 cells involved increased levels of expression of pro-apoptotic BAX mRNA and protein molecules. These data suggest that 4-MU may be a candidate therapeutic agent for the treatment of canine mammary tumors. Furthermore, this study provides the first indication that the canine mammary tumor may be a suitable model for comparative study of the function of HA in human breast cancer.